CN102212019B - Branched water-soluble polymers and their conjugates - Google Patents

Branched water-soluble polymers and their conjugates Download PDF

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CN102212019B
CN102212019B CN201110071874.2A CN201110071874A CN102212019B CN 102212019 B CN102212019 B CN 102212019B CN 201110071874 A CN201110071874 A CN 201110071874A CN 102212019 B CN102212019 B CN 102212019B
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peg
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S·德弗里斯
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Weisuofan Co Ltd
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Abstract

The present invention provides branched water-soluble polymers that allow two or more water-soluble polymers to be conjugated to another species. The branched polymers provide access to therapeutic agents that are conjugated at a single site to two or more water-soluble polymers. The branched polymers are based upon branch points that are simple branched alkyl structures, reactive side-chain amino acids and small peptides of reactive side-chain amino acids, and saccharides. Also provided is a method for preparing mono-disperse poly(ethylene glycol) of a well-defined and determinable molecular weight, and a method for the rational end-functionalization of poly(ethylene glycol). Conjugates of the branched water-soluble polymers with diverse species, e.g., peptides, lipids, glycolipids and small molecules are also provided.

Description

Branched water-soluble polymers and conjugate thereof
The cross reference of related application
The application is the US temporary patent application No.60/454 proposed on March 14th, 2003,993, the US temporary patent application No.60/474 that on May 29th, 2003 proposes, 094, and the US temporary patent application No.60/509 that on October 7th, 2003 proposes, the non-provisional of 752 is submitted to, and the content of each application is introduced for reference comprehensively herein.
Technical field
The conjugate that the present invention relates to branched water-soluble polymers and formed by these branched polymers.
Background technology
Hydrophilic polymer, such as PEG (abbreviation PEG, be also called poly-(oxyethane), abbreviation PEO) with molecule and surface be conjugated in biotechnology and field of medicaments has considerable purposes.According to its most common form, PEG is the line polymer at each end hydroxy terminal:
HO-CH 2CH 2O-(CH 2CH 2O) n-CH 2CH 2-OH
Wherein n normally about 3 to about 4000.Many end-functionalization derivatives are known in the literature, and can be commercial.Such as, see Shearwater Polymers, Inc. " Polyethylene Glycol Derivatives " catalogue.
Having not isoplastic PEG material in each of two ends is special compounds effective.Such as, Heterobifunctional PEG can be used as linking agent.And, such as use the PEG molecule of alkyl " end-blocking " (such as methoxyl group) to make the C-terminal of molecule be converted into any one of a large amount of reactive organo-functional groups at an end.
Random or the segmented copolymer of oxyethane shown below and propylene oxide is closely related with PEG in chemical structure, and they can replace PEG in many application of PEG.
HO-CH 2CHRO-(CH 2CHRO) n-CH 2CR-OH
Wherein each R is H or CH independently 3.
The formation of the conjugate between therapeutic active substance and water-soluble polymers has proved the strategy being full of effect of pharmacokinetics for improvement of therapeutical agent and pharmacodynamics.Such as, see Dunn and Ottenbrite, " Polymeric Drugs and Drug DeliverySystems ": ACS Symposium Series 469, American Chemical Society, Washington, D.C.1991.Such as, PEG is used for the verified immunogenicity also prolongation clean-up time in the circulating cycle lowering peptide of application of derivatize peptide therapeutics.Such as, US patent No.4,179,337 (people such as Davis) relate to the non-immunogenic peptide being coupled to polyoxyethylene glycol (PEG) or polypropylene glycol, such as enzyme and peptide hormone.Every mole of peptide uses 10-100 moles of polymer, and keeps the physiologically active of at least 15%.
Other examples many of PEG-peptide conjugate are well known in the art.The major way that PEG and derivative thereof are connected to peptide is the non-specific binding by peptide ammino acid residue.Such as, US patent No.4,088,538 disclose the enzymic activity polymkeric substance-enzyme conjugate being connected to the enzyme of PEG with covalent linkage.Similarly, US patent No.4,496,689 disclose the mixture that α-1 proteinase inhibitor is connected with the covalent linkage of polymkeric substance such as PEG or methoxyl group PEG (" mPEG ").(J.Biol.Chem.252:3578 (1977) discloses the covalently bound of the amido of mPEG and bovine serum albumin to the people such as Abuchowski.WO93/15189 (people such as Veronese) relates to the method keeping the activity of poly ethyldiol modified proteolytic ferment by proteolytic ferment being connected to macromole inhibitor.This conjugate object is used for medical use.US patent No.4,414,147 disclose the acid anhydrides by being puted together by Interferon, rabbit in dicarboxylic acid, such as gather (ethylene glycol Succinic anhydried) and lower its hydrophobic method.PCT WO87/00056 discloses PEG and poly-(ethoxylation) polyvalent alcohol is puted together in such as beta-interferon, the method for the albumen of interleukin-2 and immunotoxin and so on.EP 154,316 discloses and require that chemical modification lymphokine, such as contains the IL-2 of the PEG of at least one primary amino being directly bonded in this lymphokine.US patent No.4,055,635 pharmaceutical compositions disclosing the water-soluble compound being connected to the proteolytic ferment of polymeric material such as polysaccharide with covalent linkage.
Another way PEG being connected to peptide is the non-specific oxidation by the glycosyl residue on peptide.Oxidized sugar is used as site PEG structure division being connected to peptide.Such as, M ' Timkulu (WO 94/05332) discloses hydrazine-or amino-PEG for PEG being added to the purposes of glycoprotein.Glycosyl structure part is randomly oxidized to corresponding aldehyde, and they are coupled to amino-PEG subsequently.
In above-mentioned each method, PEG adds to the reactive residue on peptide backbone in random, non-specific mode.Usually, result in the loss of peptide activity with PEG derivatize, this is directly owing to the non-selective character of the chemical process for puting together this water-soluble polymers.
Another difficult problem relevant with forming conjugate between water-soluble polymers and biomolecules is the ability of reaction water-soluble polymeric reagent in more than one site-tag biomolecules.Although usually wish that each conjugate comprises more than one water-soluble polymer structure part, the decreased extent of biomolecule activity is usually directly proportional to the number of the polymer moieties being bonded in biomolecules.Therefore, obtain that per molecule comprises the reactivity of two or more water-soluble polymer structure part, branched substances is significant.By using branching molecule, more than one water-soluble polymers can be puted together in biomolecules, and without the need to the more than one site in interfere with biomolecules.
Branched polymer based on PEG is well known in the art.Such as, the people (WO 98/41562) such as Greenwald discloses the branching PEG based on DAP core.Morpurgo and colleague thereof discuss the purposes of the branching PEG based on Methionin core in Appl.Biochem.Biotechnol.56:59-72 (1996).The similar branching PEG based on Methionin by people such as Guiotto, prepared by Bioorg.Med.Chem.Lett.12:177-180 (2002).The people such as Harris (US patent No.5,932,462) have also prepared the branching PEG based on Methionin.The people such as Martinez (US patent No.5,643,575) describe the conjugate (US patent No.6,113,906) of many branching PEG materials based on various nuclear structure and these materials and biological active materials.
Polymkeric substance, such as PEG is known to be existed as heterodisperse colony, that includes polymkeric substance chain length and the molecular weight of certain limit.When preparing treatment preparation, obviously, wishes that polymkeric substance that employing has minimum heterodispersity is to guarantee consistence between preparation and circulation ratio.Be known in the art the method almost not preparing single dispersing PEG sample.The people such as Loiseau disclose the synthesis of well-defined PEG molecule.The method adopts and protects/go Preservation tactics, and this is not best for a large amount of basic single dispersing PEG of preparation.Therefore, except branching PEG polymkeric substance, prepare single dispersing PEG and be desirable especially by this single dispersing material method be incorporated in branched polymer.
The present invention has responded the demand for branched water-soluble polymers and single dispersing PEG material, opens and obtains novel therapeutic conjugate, the approach of such as peptide conjugate, and solves for more stable and treat the demand of effective therapeutant.Still demand is there is for by the method for the modified group such as industrial practicality of water-soluble polymers modification treatment biomolecules.Meaningfully wherein conjugate has the method for the performance improved than unmodified therapeutical agent especially.Present invention accomplishes these and other demand.
Summary of the present invention
The invention provides the branched water-soluble polymers of the core based on various structures.Branched polymer of the present invention provides the means by singular association position, two or more water-soluble polymer structure part being connected to another material.The present invention illustrates by reference to branched PEG molecule.PEG representatively property water-soluble polymers concentrates description to be for clarity, should not be considered to limit the present invention.Technician will appreciate that, branched substances as herein described can be prepared with any water-soluble polymers substantially.Except PEG, other Exemplary water soluble polymkeric substance comprises poly-(propylene glycol).
In first, the invention provides the branched water-soluble polymers with following general formula:
WSP-Y-R x
Wherein WSP is water-soluble polymers.Symbol Y represents connection base, such as key, or the structure division containing acid amides, carboxylicesters, urea alkane, mercaptan, replacement or non-substituted alkyl etc.Exemplary connection base comprises: key, (CH 2) n, (CH 2) mc (O) O (CH 2) n, (CH 2) mc (O) NH (CH 2) n, (CH 2) moC (O) NH (CH 2) n, (CH 2) mo (CH 2) n, (CH 2) mnH (CH 2) n, and (CH 2) ms (CH 2) n, wherein m and n is the integer independently selected from 0-6.R xbe water-soluble polymers, be connected to the replacement of water-soluble polymers or non-substituted alkyl structure division; Be connected to the amino acid of water-soluble polymers or amino acid whose dimer; Or be connected to sugar or the sugar nucleotide of water-soluble polymers.WSP and R xwater-soluble copolymer component can be identical water-soluble polymers or different water-soluble polymerss.
Exemplary water soluble polymkeric substance for compound of the present invention comprises m-PEG, PEG, m-PPG, PPG, Polysialic acid, polyglutamic acid, poly aspartic acid, polylysine, polymine, biodegradable polymkeric substance (such as polylactide, polyglycerol ester), with functionalized PEG, such as end-functionalization PEG.
In an exemplary embodiment, Y is substituted alkyl, and the invention provides the branched water-soluble polymers with following general formula:
Wherein X and Y is independently selected from OR 1, NR 2r 3, SR 4, COOR 5, CONR 6r 7, OCONR 6r 7, replace and unsubstituted alkyl, and replacement and unsubstituted aryl in member.Z 1be selected from OR 1', NR 2' R 3', SR 4', COOR 5', CONR 6' R 7', replace and non-substituted alkyl, and replace and unsubstituting aromatic yl in member.Symbol R 1, R 4and R 5represent water-soluble polymers.R 2, R 3, R 6and R 7independently selected from H, replace and non-substituted alkyl, replace and unsubstituting aromatic yl, replace and non-substituted heteroaryl, replace and unsubstituting heterocycle alkyl, reactive functional groups, and the member in water-soluble polymers, prerequisite selects these groups, makes to comprise at least two water-soluble polymer structure parts according to the compound of general formula I.Symbol R 1', R 2', R 3', R 4', R 5', R 6' and R 7' represent independently selected from H, replace and non-substituted alkyl, replace and unsubstituting aromatic yl, replace and non-substituted heteroaryl, replace and unsubstituting heterocycle alkyl, reactive functional groups, the group in carrier molecule and water-soluble polymers.
In another exemplary embodiment, Z 1comprise glycosyl (saccharyl) structure division.Glycosyl structure part can be activated glycosyl structure division, such as nucleotide sugar.In addition, Z 1the amino acid whose glycosyl structure part being directly connected in peptide can be comprised, or be indirectly connected to the amino acid whose glycosyl structure part of peptide by it with being connected to amino acid whose puting together of glycosyl residue.
Present invention also offers the branched polymer based on amino acid or oligomeric amino acid (such as dipeptides, tripeptides, tetrapeptide).Exemplary amino acid pattern branched polymer have be selected from following among general formula:
Wherein R 11, R 11', R 12, R 12', R 13' and R 13' independently selected from H, replace or non-substituted alkyl and water-soluble polymers, prerequisite selects these groups, makes above-claimed cpd comprise at least two water-soluble polymer structure parts.R 14be selected from OH, reactive functional groups, the group of sugary structure division or the one be connected in the group of carrier molecule.A is selected from NH, the one in O and S.Subscript " s " represents the integer of 1-5.
Each compound described in above general formula can be used for the chemical PEGylation of other material (such as nucleic acid, peptide, sugar etc.).The method forming the conjugate between PEG (and the material containing PEG) is well known in the art usually.Such as, see Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; With people such as Feeney, MODIFICATION OF PROTEINS; Advances in ChemistrySeries, Vol.198, American Chemical Society, Washington, D.C., 1982.
In another exemplary embodiment, R 14comprise glycosyl structure part.This glycosyl structure part can be activated glycosyl structure division, such as nucleotide sugar.In addition, R 14the amino acid whose glycosyl structure part being directly connected in peptide can be comprised, or be indirectly connected to the amino acid whose glycosyl structure part of peptide by it with being connected to amino acid whose puting together of glycosyl residue.
In yet another aspect, the invention provides the branched water-soluble polymers based on sugared core (" a type core ").Technician is clear, and sugar endorses to have any structure.Exemplary sugar for this respect of the present invention comprises GlcNAc, Gal, Sia, Fuc, Glc, Gal NAc, GalNH 2, GlcNH 2deng.
Exemplary compounds of the present invention has following general formula:
Sugar-O-(L-WSP) 2
Wherein L is connection base and WSP is water-soluble polymers.
In another exemplary embodiment, glycosyl branched water-soluble polymers of the present invention has following general formula:
Nucleotide-sugar-O-(L-WSP) 2
Another exemplary compounds (based on sialic acid core) according to this aspect of the invention has following general formula:
Wherein R 16and R 16' be selected from H, the member in ethanoyl and general formula (I):
And R 17, R 18, R 19and R 19' be independently selected from H, OH, NH 2, NHAc and according to the member in the structure division of general formula I.In general formula I, Z 2be selected from O, S, CH 2with the one in S.R 11as mentioned above, and subscript " a " represents the integer of 0-20, and prerequisite is R 16, R 16', R 17, R 18, and R 19at least two structures had according to general formula I.R 11can also be connected to the group of carrier molecule or be bonded in the key of carrier molecule.R 15be selected from H and activating group, such as, a member in phosphoric acid nucleoside acid.
In yet another aspect, this branched polymer is based on semi-lactosi or N-acetyl galactose, and it has following general formula:
Wherein R 15-R 19as mentioned above, R 15-R 19at least two be structure division according to general formula I.
The structure that other exemplary sugar with general formula as implied above derives is seminose and the branched water-soluble polymers based on glucose.
In addition, R 15can comprise the amino acid whose key being connected to peptide, or be connected to the key of glycosyl structure part, this glycosyl structure part is bonded directly to the amino acid of peptide, or by put together in connection amino acid whose glycosyl residue and indirect linkage in the amino acid of peptide.
Present invention also offers the method for the basic single dispersing colony of preparing PEG molecule.The method comprises the PEG molecule allowing and have clear and definite molecular weight, the difunctionality activated PEG also with clear and definite molecular weight of such as PEG200 and at least 2 equivalents, such as PEG200 contact, thus produces the single dispersing sample of PEG, such as PEG 600:
G is leavings group, such as sulphonate or tresylate ester.Then the single dispersing sample of PEG600 can contact with difunctionality activated PEG 200, forms single dispersing PPEG100.Or single dispersing PEG600 can be converted into corresponding Bifunctionalized derivative, then react with the single dispersing dihydroxyl-PEG600 of at least 2 equivalents, produce single dispersing PEG1800.Repeat method of the present invention, until obtain the single dispersing PEG of required size.This synthesis can be designed, make the molecular weight differences between starting raw material and product allow to be separated by size exclusion chromatography, the material of any unreacted or partial reaction.
And as to preparing modified water-soluble polymer, the response of the demand of improving one's methods of such as PEG, the invention provides chemical activation and extends the method for polymer backbone.Single activated PEG molecules can be used for being puted together by PEG in various material, such as target structure division, treatment structure division, antitumor drug, cytotoxin, radiological agent, amino acids, sugar etc.
Therefore, in yet another aspect, the invention provides activated water-soluble polymer, the method for the especially substep assembling of PEG and analog thereof.This method provide obtain monofunctional and Bifunctionalized PEG molecule facilitate approach.
Therefore, in an exemplary embodiment, the invention provides the method preparing PEG.Outline the method below:
A.R-Y/ (acid or alkali); B. activate, such as tosylation, halogen-go hydroxylation, such as HX or SOX 2, and and PEG mreaction; C. activate (R '), such as, use chloroformic acid p-nitrophenyl ester.
Wherein, subscript m and n represent 1-100 independently, 000.
In step a, original glycol contacts with activating group (R-Y), and the hydroxyl moiety of this activating group and glycol reacts.Y is leavings group normally, and R is placed on one of hydroxyl moiety of PEG molecule.In stepb, the group that the free hydroxyl group of gained adducts is converted into sulfonic acid esters by it activates.Activation PEG material with as initial PEG (" PEG m") the identical or different polymerization degree another PEG structure division contact.Another material is connected to, RO-PEG in order to make it (n+m)optionally activate in free hydroxyl group structural portion office.
Compound of the present invention can be used for by the direct chemical PEGylation of one or more obtainable reactive residue on therapeutical agent, forms substrate such as therapeutical agent, such as peptide, lipid, the water-soluble polymers conjugate of glycolipid.Compound of the present invention is also easily incorporated into can at substrate, in the glycoconjugate of such as, in the sugar-PEGization of the enzyme mediation of the therapeutical agent activation of employing.
Present invention also offers the pharmaceutical preparation that one or more branched water-soluble polymers of the present invention puts together therapeutical agent thereon.Additionally provide the method for disease therapy, these diseases are improved by the conjugate given between therapeutical agent and branched water-soluble polymers of the present invention or are cured.
Other side of the present invention, advantage and object can be recognized from following detailed description.
Accompanying drawing is sketched
Detailed description of the present invention
Abbreviation
PEG, PEG; M-PEG, methoxyl group-PEG; PPG, poly-(propylene glycol); M-PPG, methoxyl group-poly-(propylene glycol); Fuc, fucosido; Gal, galactosyl; GalNAc, N-acetylamino galactosamine base; Glc, glucosyl group; GlcNAc, NAG base; Man, epichitosamine base; ManAc, epichitosamine yl acetate; Sia, sialic acid; And the neural amido (N-acetylneuraminyl) of NeuAc, N-ethanoyl.
Definition
Unless otherwise defined, whole technology used herein has the implication identical with the general understanding of those skilled in the art usually with scientific terminology.Usually, herein and the nomenclature used in the experimental procedure of cell cultures, molecular genetics, organic chemistry and nucleic acid chemistry and cross discipline be well-known and this area commonly use those.Nucleic acid and peptide symthesis adopt standard technique.This technology and operation generally according to the usual way of this area and various general reference carry out (generally see, the people such as Sambrook, MOLECULAR CLONING; A LABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., the document is incorporated herein for reference), they are provided in whole file.Herein and the nomenclature adopted in the experimental procedure of analytical chemistry and following organic synthesis be well-known and conventional those in this area.Chemosynthesis and chemical analysis adopt standard technique or their improvement.
What term as used herein " sugar is puted together (glycoconjugation) " referred to that the amino acid of modified sugars material and peptide or the enzyme of saccharide residue mediate puts together.The subgenus of " sugar is puted together " is " sugar-PEGization ", and wherein the modified group of modified sugars is PEG, and its alkyl derivative (such as m-PEG) or reactive derivatives (such as H 2n-PEG, HOOC-PEG).
Term " sialic acid " refers to any member of the family of the carboxylated sugar of nine carbon.The modal member of sialic acid family is NeuAc (2-ketone-5-acetylaminohydroxyphenylarsonic acid 3; 5-dideoxy-D-glycero-D-galacto ketononose pyranose-1-acid (galactononulopyranos-1-onic acid); often be abbreviated as Neu5Ac, NeuAc or NANA).Second member of this family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), and wherein the N-ethanoyl of NeuAc is by hydroxylation.3rd sialic acid family member is 2-ketone-3-deoxidation-nonanone saccharic acid (nonulosonic acid) (KDN) (people (1986) such as Nadano, J.Biol.Chem.261:11550-11557; The people such as Kanamori, J.Biol.Chem.265:21811-21819 (1990)).Further comprises the sialic acid such as 9-O-C that 9-replaces 1-C 6acyl group-Neu5Ac is as 9-O-lactoyl-Neu5Ac or fluoro-Neu5Ac and the 9-azido-of 9-O-ethanoyl-Neu5Ac, 9-deoxidation-9--9-'-deoxy-n eu5Ac.About the commentary of sialic acid family, for example, see Varki, Glycobiology 2:25-40 (1992); Sialic Acids:Chemistry, Metabolism and Function, R.Schauer, Ed. (Springer-Verlag, New York (1992)).The synthesis of sialic acid compound in sialylated operation and purposes are disclosed in the international application WO92/16640 published on October 1st, 1992.
" peptide " refers to polymkeric substance, and wherein monomer is amino acid and is connected to by amido linkage-is risen, or is referred to as polypeptide.In addition, alpha-non-natural amino acid, such as Beta-alanine, phenylglycocoll and homoarginine are also included.The amino acid of non-genomic coding also can adopt in the present invention.In addition, be modified into and comprised reactive group, glycosylation site, polymkeric substance, treatment structure division, the amino acid of biomolecules etc. also can adopt in the present invention.The all amino acid used in the present invention can be D-or L-isomer.L-isomer is generally preferred.In addition, other plan peptide (peptidomimetics) also can be used for the present invention." peptide " used herein refers to glycosylated peptide and non-glycosylated peptide.Further comprises the incomplete glycosylated peptide of the system being expressed peptide.About general commentary, see Spatola, A.F., CHEMISTRY ANDBIOCHEMISTRY OF AMINO ACIDS, PEPTIDES AND PROTEINS, B.Weinstein, eds., Marcel Dekker, New York, p.267 (1983).
Term " peptide conjugate " refers to material of the present invention, and wherein peptide and modified sugars as described herein are carried out sugar and puted together.Representatively property example, this peptide is the mutant peptide with the glycosylation site that the O-be not present in wild type peptide connects.
Term " amino acid " refers to the amino acid of natural existence and synthesis, and amino acid analogue and with the plan amino acid worked with mode like naturally occurring amino acids.Naturally occurring amino acid is those amino acid coded by genetic code, and those amino acid of modification afterwards, such as oxyproline, Gla, and O-phosphoserine.Amino acid analogue refers to the compound with the basic chemical structure identical with naturally occurring amino acid, that is, have and be bonded in hydrogen, carboxyl, the α carbon of amino and R group, such as homoserine, nor-leucine, methionine sulphoxide, methionine(Met) methyl sulfonium.These analogues have modification R group (such as nor-leucine) or modification peptide backbone, but remain the basic chemical structure identical with naturally occurring amino acid.Intend amino acid and refer to the structure having and be different from amino acid whose general chemical structure, but with the compound worked with mode like naturally occurring amino acids.
As used herein term " modified sugars " refers to the carbohydrate that natural existence or non-natural exist, with branched water-soluble polymers modification of the present invention, and can on the enzymatic amino acid that adds to peptide, lipid, glycolipid etc. or glycosyl residue.Modified sugars is selected from many enzyme substratess, including, but not limited to sugar nucleotide (Monophosphate, bisphosphate and triguaiacyl phosphate), and activation sugar (such as halogen-sugar, glycosyl methanesulfonates) and both do not activated the carbohydrate of also non-nucleotidylation." modified sugars " is with " modified group " (it is branched polymer of the present invention) covalent functionalization.The position that selection modified group is functionalized, makes it not hinder " modified sugars " enzymatic to add on peptide or other substrate.
Term " water-soluble " refers to the structure division in water with the solubleness that can detect.Detection and/or quantitative water miscible method are well-known in the art.Exemplary water soluble polymkeric substance comprises peptide, carbohydrate, poly-(ether), poly-(amine), poly-(carboxylic acid) etc.Peptide can have the mixed sequence be made up of single amino acid, such as poly-(Methionin).Exemplary polysaccharide is poly-(sialic acid).Exemplary poly-(ether) is PEG, such as m-PEG.Poly-(ethyleneimine) is exemplary polyamine, and poly-(vinylformic acid) is representative poly-(carboxylic acid).
Term " PEG ", " PEG ", " poly-(propylene glycol) " and " PPG " use with their general significance, and they further comprises the derivative of this parent compound, such as monoalkyl material, such as m-PEG, m-PPG, reactive materials, N-hydroxysuccinimide, p-nitrophenyl carbonate (p-NP), HOBT derivative, and amine.Also be included within the scope of these terms is the material comprising two or more modification, such as p-NP-PEG-OMe etc.
Term as used herein " glycosyl connection base " refers to glycosyl residue, and agent (such as water-soluble polymers, treatment structure division, biomolecules) is connected to it with covalent linkage.In the method for the invention, " glycosyl connection base " is connected to glycosylation or non-glycosylated peptide with covalent linkage, thus on amino acid agent is connected on peptide and/or glycosyl residue." glycosyl connection base " is generally connected with the amino acid of peptide and/or the enzymatic of glycosyl residue by " modified sugars " and is produced by " modified sugars "." complete glycosyl connect base " refers to the connection base derived by glycosyl structure part, and each sugar monomer wherein connecting conjugate is not degraded, such as not oxidized, such as not be oxidized by sodium metaperiodate." intact glycosyl connection base " of the present invention can by increasing glycosyl units or removing one or more glycosyl units and produced by naturally occurring oligosaccharides from parent sugar structure.
" pharmaceutically acceptable carrier " used herein comprises any material, when with conjugate in conjunction with time, maintain the activity of conjugate, and not with the immune system response of patient.Example is including, but not limited to any one of standard pharmaceutical carriers, and the salt brine solution of such as phosphate buffered, water, emulsion is oil/water emulsion such as, and various types of wetting agent.Other carriers can also comprise thimerosal, tablet, comprise coated tablet and capsule.Typically, these carriers contain excipient such as starch, milk, sugar, the clay of some type, gelatin, stearic acid or its salt, Magnesium Stearate or calcium stearate, talcum, vegetation fat or oil, natural gum, glycols, or other known excipient.Examples of such carriers can also comprise flavouring agent and color additives or other composition.The composition comprising examples of such carriers is prepared by known usual way.
" administration " used herein refers to oral administration, as suppository dosing, and localized contact, intravenously, intraperitoneal, intramuscular, damage zone, or subcutaneous administration, inhalation, or the eluting device in patients with implantation body, such as miniosmotic pump.Administration, by any approach, comprises non-bowel and through mucous membrane mode (such as oral, intranasal, transvaginal, per rectum or transdermal), particularly by suction.Parenterai administration comprises such as intravenously, intramuscular, in arteriole, and intradermal, subcutaneous, intraperitoneal, in ventricle, and encephalic.And, if injection be used for the treatment of tumour, such as, bring out apoptosis, can directly administration to the tissue around tumour and/or tumour.Other mode of movement is including, but not limited to the agent of use liposome formulation, and intravenously inputs, transdermal patch etc.
Term " separation " refers to substantially or is substantially free of the material of the component for the production of this material.For peptide conjugate of the present invention, term " separation " refer to substantially or be substantially free of normally in for the preparation of the mixture of peptide conjugate this material with the material of component." separation " and " pure " can exchange use.Typically, the peptide conjugate of separation of the present invention has preferably by the certain purity of Range Representation.The lower limit of peptide conjugate purity range is about 60%, about 70% or about 80%, and the upper limit of purity range is about 70%, about 80%, about 90% or exceed about 90%.
When peptide conjugate exceedes about 90% purity, their purity also preferably presses Range Representation.The lower limit of purity range is about 90%, about 92%, about 94%, about 96%, or about 98%.The upper limit of purity range is about 92%, about 94%, about 96%, about 98% or about 100% purity.
The analytical procedure that purity is generally acknowledged by any technology measures (such as, the band intensity on the gel of Silver stain, polyacrylamide gel electrophoresis, HPLC or similar fashion).
" substantially each member of colony " used herein describes the characteristic of the colony of peptide conjugate of the present invention, wherein the percentile modified sugars be added on polypeptide selected is added on the multiple identical acceptor site on peptide." substantially each member of colony " has mentioned " homogeneity " in the site of puting together the peptide in modified sugars, refers at least about 80%, and preferably at least about 90% and more preferably at least about 95% uniform conjugate of the present invention.
" homogeneity " refers to that modified sugars puts together the structural integrity of the colony of receptor structure part thereon.Therefore, each modified sugars structures moiety conjugation is in the peptide conjugate of the present invention of the structure acceptor site identical with the acceptor site that other modified sugars each are puted together wherein, and this peptide conjugate is said to be about 100% uniform.Homogeneity represents by scope usually.The lower limit of the homogeneity scope of peptide conjugate is about 60%, about 70%, or about 80%, and the upper limit of purity range is about 70%, about 80%, about 90%, or exceed about 90%.
When peptide conjugate exceedes or equal about 90% homogeneity, their homogeneity also preferably presses Range Representation.The lower limit of the scope of homogeneity is about 90%, about 92%, about 94%, about 96% or about 98%.The upper limit of purity range is about 92%, about 94%, about 96%, about 98% or about 100% homogeneity.The purity of peptide conjugate generally by one or more methods well known by persons skilled in the art, such as C/MS (liquid chromatography-mass spectrography) (LC-MS), substance assistant laser desorpted time-of-fight mass spectrometry (MALDITOF), capillary electrophoresis etc.
When mentioning glycopeptide material, " basic uniform sugared shape " or " basic uniform glycoforms " refers to by the percentage of the glycosylated receptor structure part of the glycosyltransferase being studied (such as fucosyltransferase).Such as, when α 1,2-fucosyltransferase, if substantially own the Gal β 1 of (as defined hereinafter) in peptide conjugate of the present invention, 4-GlcNAc-R and the acid-group analogue of saliva thereof by fucosylation, then have basic uniform fucosylated form.It will be apparent to those skilled in that, starting raw material can contain glycosylation acceptor structure division (such as fucosylation Gal β Isosorbide-5-Nitrae-GlcNAc-R structure division).Therefore, the glycosylation percentage of calculating comprises by the glycosylated receptor structure part of method of the present invention, and those receptor structure parts glycosylated in starting raw material.
Term " substantially " in the above definition of " substantially even " generally refers at least about 40% of specific glycosyltransferase, at least about 70%, at least about 80%, or more preferably at least about 90%, also more preferably the receptor structure part of at least about 95% is glycosylated.
Term " on a large scale " and " technical scale " are used interchangeably, and refer at the end of a reaction time, to create at least approximately 250mg, preferably the reaction time of at least approximately 500mg and the more preferably glycoconjugate of at least about 1g.
No matter be used as key or show vertical with key, symbol structure division shown by expression is connected to the remainder of molecule, the position of solid carrier etc.
Some compound of the present invention can exist with nonsolvated forms and solvation form, comprises hydrated form.Generally, solvation form is equal to nonsolvated forms, and comprises within the scope of the invention.Some compound of the present invention can exist with multiple crystallization or amorphous form.Generally, for the application contemplated by the present invention, all physical form are equivalent, and are within the scope of the invention.
Some compound of the present invention has asymmetric c atom (optical center) or double bond; Racemic modification, diastereomer, geometrical isomer and individual isomer comprise within the scope of the invention.
Compound of the present invention can as individual isomer (such as enantiomorph, cis-trans, position, diastereomer) or the mixture as isomer.In a preferred embodiment, compound is as individual isomer preparation substantially.The method preparing isomery pure compound is substantially well known in the art.Such as, the mixture and the pure enantiomeric compounds that are rich in enantiomorph can be prepared with keeping the stereochemistry reaction that is constant or that cause it to change completely of chiral centre by using the synthetic intermediate of pure enantiomorph.Or the intermediate in final product or route of synthesis can be resolved as single stereoisomers.Conversion or keep the constant technology of specific Stereocenter and be known in the art for those technology of the mixture of resolving steric isomer, and be obviously that those skilled in the art selects appropriate means in the limit of power of particular case.Usually, can see the people such as Furniss (ed s.), VOGEL ' SENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5 tHeD., LongmanScientific and Technical Ltd., Essex, 1991, pp.809-816; And Heller, Acc.Chem.Res.23:128 (1990).
Compound of the present invention can also contain the atom isotope of non-natural ratio at the one or more atom places forming this compounds.Such as, these compounds can use radio isotope, such as tritium ( 3h), iodine-125 ( 125i) or carbon-14 ( 14c) mark.No matter whether have radioactivity, all isotopic variations intentions of compound of the present invention comprise within the scope of the invention.
If substituting group their generic chemical formulae from left to right write represents, they include by the chemically uniform substituting group write structural formula from right to left and obtain equally, such as-CH 2o-can also be written as-OCH 2-.
Unless otherwise prescribed, otherwise itself or the term " alkyl " as another substituent part refer to straight or branched, or cyclic hydrocarbon group, or their binding substances, it can be entirely saturated, single or polyunsaturated, divalence (" alkylidene group ") and multivalence group can be comprised, there is carbonatoms (the i.e. C specified 1-C 10refer to 1-10 carbon atom).The example of saturated hydrocarbyl is including, but not limited to such as following and so on group: methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, the tertiary butyl, isobutyl-, sec-butyl, cyclohexyl, (cyclohexyl) methyl, Cvclopropvlmethvl, analogue and isomer, such as n-pentyl, n-hexyl, n-heptyl, the analogue of n-octyl etc. and isomer.Unsaturated alkyl is the alkyl with one or more double bond or triple bond.The example of unsaturated alkyl including, but not limited to vinyl, 2-propenyl, crot(on)yl, 2-isopentene group, 2-(butadienyl), 2,4-pentadienyl, 3-(Isosorbide-5-Nitrae-pentadienyl), ethynyl, 1-and 3-proyl, 3-butynyl, and senior analogue and isomer.Unless otherwise defined, term " alkyl " is also intended to those derivatives of the alkyl being included in following more specific definition, such as " assorted alkyl ".The alkyl being limited to alkyl is called as " same to alkyl (homoalkyl) ".
About 1 is contained to about 25 carbon atoms (such as methyl, ethyl etc.) for exemplary alkyl of the present invention.There is≤the straight chain of 8 carbon atoms, branching or cyclic hydrocarbon chain will be referred to herein as " low alkyl group ".In addition, term as used herein " alkyl " is included in the one or more substituting groups on one or more carbon atoms of hydrocarbon chain fragment further.
Term " alkoxyl group ", " alkylamino " or " alkylthio " (or thio alkoxy) uses with their ordinary meaning, refers to those alkyl being connected to the remainder of molecule respectively via Sauerstoffatom, amino or sulphur atom.
Unless otherwise prescribed, itself or the term " assorted alkyl " be combined with another term refer to straight or branched, or ring-type carbon-containing group, or their binding substances, be made up of with at least one heteroatoms be selected from O, N, Si, P and S the carbon atom of described number, wherein nitrogen, p and s atom are optionally oxidized, and nitrogen heteroatom is optionally quaternary.Heteroatoms O, N, P, S and Si can be positioned at the position that any interior location of assorted alkyl or alkyl are connected to the remainder of molecule.Example is including, but not limited to-CH 2-CH 2-O-CH 3,-CH 2-CH 2-NH-CH 3,-CH 2-CH 2-N (CH 3)-CH 3,-CH 2-S-CH 2-CH 3,-CH 2-CH 2-S (O)-CH 3,-CH 2-CH 2-S (O) 2-CH 3,-CH=CH-O-CH 3,-Si (CH 3) 3,-CH 2-CH=N-OCH 3, and-CH=CH-N (CH 3)-CH 3.Two heteroatomss can be continuous print at the most, such as, and-CH 2-NH-OCH 3with-CH 2-O-Si (CH 3) 3.Similarly, itself or the term " sub-assorted alkyl " as another substituent part refer to by the divalent group of assorted alkyl derivative, such as but be not limited to-CH 2-CH 2-S-CH 2-CH 2-and-CH 2-S-CH 2-CH 2-NH-CH 2-.To mix alkyl for Asia, heteroatoms can also occupy chain end one or two (such as alkylene oxide group, alkylenedioxy group, alkylidene amino, alkylenediamino etc.).In addition, be connected base for alkylidene group and Asia alkyl of mixing, the No yield point connecting base represents with the direction that the chemical formula wherein connecting base is write.Such as chemical formula-C (O) 2r '-expression-C (O) 2r '-and-R ' C (O) 2-.
Unless otherwise prescribed, itself or the term " cycloalkyl " that is combined with other term and " Heterocyclylalkyl " represent the ring-type modification of " alkyl " and " alkyl of mixing " respectively.In addition, for Heterocyclylalkyl, heteroatoms can occupy the position that heterocycle is connected to the remainder of molecule.The example of cycloalkyl including, but not limited to cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, suberyl etc.The example of Heterocyclylalkyl is including, but not limited to 1-(1,2,5,6-tetrahydro pyridyl), piperidino, 2-piperidyl, 3-piperidyl, 4-morpholinyl, morpholinyl, tetrahydrofuran (THF)-2-base, tetrahydrofuran (THF)-3-base, tetramethylene sulfide-2-base, tetramethylene sulfide-3-base, 1-piperazinyl, 2-piperazinyl etc.
Unless otherwise prescribed, term " aryl " refers to how unsaturated Aromatic moieties, and it can be monocycle or many rings (a preferred 1-3 ring), condenses together or covalent linkage connection.Term " heteroaryl " refers to that wherein nitrogen and sulphur atom are optionally oxidized, and nitrogen-atoms is optionally quaternary containing the 1-4 be selected from N, O and S heteroatomic aryl (or ring).Heteroaryl can be connected to the remainder of molecule by heteroatoms.The limiting examples of aryl and heteroaryl comprises phenyl, 1-naphthyl, 2-naphthyl, 4-xenyl, 1-pyrryl, 2-pyrryl, 3-pyrryl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2- azoles base, 4- azoles base, 2-phenyl-4- azoles base, 5- azoles base, 3-is different azoles base, 4-is different azoles base, 5-is different azoles base, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purine radicals, 2-benzimidazolyl-, 5-indyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, tetrazyl, benzo [b] furyl, benzo [b] thienyl, 2,3-dihydrobenzo [Isosorbide-5-Nitrae] two piperazine-6-base, benzo [1,3]-dioxole-5-base and 6-quinolyl.Above-mentioned aryl and heteroaryl ring system substituting group is separately selected from following acceptable substituting group.
In order to for simplicity, when being combined with other term (such as aryloxy, fragrant sulphur oxygen base (arylthioxyl), aralkyl), term " aryl " comprises as aryl defined above and heteroaryl.Therefore, term " aralkyl " comprises those groups (such as benzyl that wherein aryl is connected to alkyl, styroyl, pyridylmethyl etc.), comprise those alkyl (such as phenoxymethyl that in described alkyl, carbon atom (such as methylene radical) is replaced by such as Sauerstoffatom, 2-pyridyloxy methyl, 3-(1-naphthyloxy) propyl group etc.).
Each term (such as " alkyl ", " assorted alkyl ", " aryl " and " heteroaryl ") comprises the replacement of described group and does not replace form above.The following provide the preferred substituting group of all kinds of group.
The substituting group of alkyl and assorted alkyl (comprise and be usually called alkylidene group, alkenyl, sub-assorted alkyl, Heteroalkenyl, alkynyl, cycloalkyl, Heterocyclylalkyl, cycloalkenyl group and heterocycloalkenyl) is commonly referred to as " alkyl substituent ", they can be selected from but be not limited to the one or more of the various groups in following group :-OR ',=O,=NR ',=N-OR ',-NR ' R " ;-SR ' ;-halogen ;-SiR ' R " R ' " ;-OC (O) R ' ,-C (O) R ' ,-CO 2r ' ,-CONR ' R " ,-OC (O) NR ' R " ,-NR " C (O) R ' ,-NR '-C (O) NR " R ' " ,-NR " C (O) 2r ' ,-NR-C (NR ' R " R ' ")=NR " " ,-NR-C (NR ' R ")=NR ' " ,-S (O) R ' ,-S (O) 2r ' ,-S (O) 2nR ' R " ,-NRSO 2r ' ,-CN and-NO 2, number is 0 to (2m '+1), and wherein m ' is the sum of the carbon atom in this group.R ', R ", R ' " and R " " preferably independently represents hydrogen separately, replaces or do not replace assorted alkyl, replace or unsubstituting aromatic yl, such as, by the aryl of 1-3 halogen substiuted, replace or non-substituted alkyl, alkoxyl group or thio alkoxy, or aralkyl.Such as, when compound of the present invention comprises more than one R group, when there are these groups more than one, each R group is selected independently, each R ', R ", R ' " and R " " select equally independently by group.As R ' and R, " when being connected to same nitrogen-atoms, they can be combined with this nitrogen-atoms, form 5,6 or 7 rings.Such as ,-NR ' R " is intended to including, but not limited to 1-pyrrolidyl and 4-morpholinyl.Discuss more than substituent, it will be apparent to those skilled in that, term " alkyl " is intended to the group comprised containing the group being bonded in non-hydrogen base, such as haloalkyl (such as-CF 3with-CH 2cF 3) and acyl group (such as ,-C (O) CH 3,-C (O) CF 3,-C (O) CH 2oCH 3deng).
With similar for the substituting group described in alkyl, the substituting group of aryl and heteroaryl is commonly referred to as " aryl substituent ".These substituting groups are such as selected from: halogen ,-OR ' ,=O ,=NR ' ,=N-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-SiR ' R " R ' " ,-OC (O) R ' ,-C (O) R ' ,-CO 2r ' ,-CONR ' R " ,-OC (O) NR ' R " ,-NR " C (O) R ' ,-NR '-C (O) NR " R ' " ,-NR " C (O) 2r ' ,-NR-C (NR ' R " R ' ")=NR " " ,-NR-C (NR ' R ")=NR ' " ,-S (O) R ' ,-S (O) 2r ' ,-S (O) 2nR ' R " ,-NRSO 2r ' ,-CN and-NO 2,-R ' ,-N 3,-CH (Ph) 2, fluorine (C 1-C 4) alkoxyl group, and fluorine (C 1-C 4) alkyl, number is 0 to the valent sum of the opening on aromatic ring system; Wherein R ', R ", R " " and R " " are preferential independently selected from hydrogen, replace or non-substituted alkyl, replace or do not replace assorted alkyl, replace or unsubstituting aromatic yl and replacement or non-substituted heteroaryl.Such as, when compound of the present invention comprises more than one R group, when there are these groups more than one, each R group is selected independently, each R ', R ", R ' " and R " " select equally independently by group.In following route map, sign X " R " as above.
Two substituting groups on the adjacent atom of aryl or heteroaryl can optionally by chemical formula-T-C (O)-(CRR ') qthe substituting group of-U-replaces, and wherein T and U is-NR-,-O-,-CRR '-or singly-bound independently, and q is the integer of 0-3.Or two substituting groups on the adjacent atom of aryl or heteroaryl ring can optionally by chemical formula-A-(CH 2) rthe substituting group of-B-replaces, wherein A and B be independently-CRR '-,-O-,-NR-,-S-,-S (O)-,-S (O) 2-,-S (O) 2nR '-or singly-bound, and r is the integer of 1-4.One of singly-bound of the new ring of such formation can optionally be replaced by double bond.Or two substituting groups on the adjacent atom of aryl or heteroaryl ring can optionally by chemical formula (CRR ') s-X-(CR " R ' ") d-substituting group replace, wherein s and d is the integer of 0-3 independently, and X be-O-,-NR '-,-S-,-S (O)-,-S (O) 2-or-S (O) 2nR '-.Substituent R, R ', R " and R ' " are preferably independently selected from hydrogen or replacement or do not replace (C 1-C 6) alkyl.
Term as used herein " heteroatoms " comprises oxygen (O), nitrogen (N), sulphur (S), phosphorus (P) and silicon (Si).
Term " amino " or " amido " refer to group-NR ' R " (or-N+RR ' R "), wherein R, R ' and R " independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heteroaryl and substituted heteroaryl.The amine replaced is that wherein R ' or R " is not the amido of hydrogen.In primary amino, R ' and R " being all hydrogen, and in secondary amino group, R ' or R " any one (but not being two) is hydrogen.In addition, term " amine " and " amino " can comprise the protonated of nitrogen and quaternary modification, comprise group-N+RR ' R " and its biocompatibility anionic counter-ion.
Unless otherwise prescribed, itself or refer to fluorine, chlorine, bromine or iodine atom as the term " halo " of another substituent part or " halogen ".Term such as " haloalkyl " intention comprises single haloalkyl and multi-haloalkyl in addition.Such as, term " halo (C 1-C 4) alkyl intention including, but not limited to trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl etc.
Term as used herein " connection base " or " L " refer to the single covalent linkage or serial stable covalent linkage that include 1-20 the non-hydrogen atom be selected from C, N, O, S and P, water-soluble polymers or branched water-soluble polymers be connected to another structure division such as chemically reactive group or put together material with covalent linkage, comprise biology and abitotic substance.Exemplary connection portion comprises the structure division containing-C (O) NH-,-C (O) O-,-NH-,-S-,-O-etc." the connection base of cleavable " be have can by the result of reacting or condition the connection base of one or more cleavable moiety that ruptures.Term " group of cleavable " refers to and connects the key of the remainder of structure division and the conjugate that will slough by cracking and make the structure division that a part for conjugate such as water-soluble polymers is sloughed from the remainder of conjugate.This cracking is chemical property, or enzyme mediation.Exemplary enzymatic lysis group comprises natural amino acid or the peptide sequence by natural amino acid terminal.
Except enzymatic lysis group, comprise one or more can by the site of the effect of non-enzymatic agent institute cracking.Exemplary non-enzymatic cracking agent including, but not limited to acid, alkali, light (such as nitrobenzyl derivatives, phenacyl, benzoin esters) and heat.Many cleavable moiety are well known in the art.For example, see people such as Jung, Biochem.Biophys.Acta, 761:152-162 (1983); The people such as Joshi, J.Biol.Chem., 265:14518-14525 (1990); The people such as Zarling, J.Immunol., 124:913-920 (1980); The people such as Bouizar, Eur.J.Biochem., 155:141-147 (1986); The people such as Park, J.Biol.Chem., 261:205-210 (1986); The people such as Browning, J.Immunol., 143:1859-1867 (1989).And difunctionality (with difunctionality and the Heterobifunctional) spacerarm (spacer arm) of multiple cleavable can be commercial.
Exemplary cleavable moiety ester is can by the cleavable moiety of reagent such as sodium hydroxide cracking, obtains the product of fragment containing carboxylicesters and hydroxyl.
This connection base can be used for this compound to be connected to another component of conjugate, such as targeting moiety (such as antibody, part, Non-covalent protein conjugated group etc.), analyte, biomolecules, medicine etc.
" Non-covalent protein conjugated group " is with the interactional structure division of associated form with complete or denatured polypeptide.This interaction can be reversible or irreversible in coenocorrelation." Non-covalent protein conjugated group " is incorporated in fluorescent chemicals of the present invention the compound providing and have with non-covalent fashion and the interactional ability of polypeptide.Exemplary noncovalent interaction comprises hydrophobic-hydrophobic and electrostatic interaction.Exemplary " Non-covalent protein conjugated group " comprises anionic group, such as phosphate radical, thiophosphoric acid root, phosphonate radical, carboxylate radical, borate, sulfate radical, sulfone, thiosulfate anion, and thiosulfonic acid root.
" nucleic acid " used herein refers to DNA, RNA, strand, the hybridization motif of double-strand or more high aggregation, and their any chemical variant.Variant is including, but not limited to providing those that additional charge, polarizability, hydrogen bonding, electrostatic interaction and three-dimensional volatility (fluxionality) be incorporated into the chemical group of nucleic acid ligands main body or whole nucleic acid ligands.This type of variant is including, but not limited to peptide nucleic acid(PNA) (PNA), phosphodiester variant (such as thiophosphatephosphorothioate, methylphosphonate), 2 '-position sugar variant, 5-position pyrimidine variant, 8-position purine variant, the outer amine variant of ring, the replacement of 4-thiouridine, the replacement of 5-bromine or the iodo-uridylic of 5-; Backbone modification, methylates, improper base-pairing combinations such as isobase, different cytidine and different guanidine etc.Nucleic acid can also comprise nonnatural base, such as nitroindoline.Variant can also comprise 3 ' and 5 ' variant such as use quencher, fluorophore or other moeites.
Term as used herein " reactive group " refers to and can react to be formed the group of covalent linkage with other chemical group, that is, under appropriate reaction conditions can the group of covalent reaction, and usually constitutes the link position of other material.This reactive group be on compound of the present invention can from the functional group reactions on different compound to form covalent linkage, obtain the structure division of fluorescence or fluorescent mark component, such as carboxylic acid or succimide ester.Reactive group generally includes nucleophile, electrophile and photoactivation group.
Exemplary reaction group comprises, but be not limited to alkene, alkynes, alcohol, phenol, ether, oxide compound, halogenide, aldehyde, ketone, carboxylic acid, ester, acid amides, cyanate, isocyanic ester, thiocyanic ester, lsothiocyanates, amine, hydrazine, hydrazone, hydrazides, diazonium, diazonium salt, nitro, nitrile, mercaptan, sulfide, disulphide, sulfoxide, sulfone, sulfonic acid,-sulfinic acid, acetal, ketone acetal, acid anhydrides, sulfuric ester, sulfenic acid, isonitrile, amidine, imide, imido-ester, nitrone, azanol, oxime, hydroxamic acid, sulfo-hydroxamic acid, propadiene class, ortho ester, sulfite, enamine, ynamine, ureas, pseudo-urea class, Urea,amino-, Carbodiimides, carbamate, imines, trinitride, azo-compound, azoxy compound, and nitroso compound.Reactive functional groups also comprises for the preparation of those of bioconjugates, such as N-hydroxysuccinimide ester, maleimide etc.The method preparing each of these functional groups is known in the art.They are (for example, see Sandler and Karo in the limit of power of those skilled in the art for the application of specific purpose or the change made in order to specific purpose, eds.ORGANIC FUNCTIONAL GROUP PREPARATIONS, Academic Press, SanDiego, 1989).
Term " target group " refers to entity (such as fluorescence structure part) vector region, such as cell that it can connect by (1) on one's own initiative; Or the structure division that (2) are preferably adsorbed by target area passively or carried secretly in target area.Target group can be small molecules, and expection comprises non-peptide and peptide.Target group can also be polymer, and including, but not limited to carbohydrate, lectin, acceptor, the part of acceptor, protein ratio is as BSA, and antibody, poly-(ether), dendritic macromole, gathers (amino acid) etc.
" carrier molecule " used herein refers to any molecule connecting compound of the present invention.Representative carrier molecule does not comprise albumen (such as enzyme, antibody), glycoprotein with not being with restriction, peptide, sugar (such as monose, oligosaccharides and polysaccharide), hormone, acceptor, antigen, substrate, metabolite, transition state analog, cofactor, inhibitor, medicine, dyestuff, nutrition, somatomedin etc." carrier molecule " also expresses possibility the material be not considered in the classical range of definition of " molecule ", such as solid carrier (such as synthetic vectors, chromosorb, film), virus and microorganism.
Preamble
The invention provides the conjugate of branched water-soluble polymers and branched water-soluble polymers.This conjugate is formed at branched water-soluble polymers of the present invention and comprising between material that this branched water-soluble polymers can put together reactive group thereon.Exemplary conjugate counterpart for water-soluble polymers of the present invention comprises peptide, glycopeptide, lipid and glycolipid.Exemplary conjugate is that the modified sugars direct or indirect (such as by getting involved glycosyl residue) of wherein carrying branched water-soluble polymers of the present invention is connected on the glycosylation site of peptide.Additionally provide the method for producing conjugate of the present invention.
The method of conjugate of the present invention and formation conjugate illustrates with reference to peptide and glycopeptide conjugate here.The focus discussed is to clearly demonstrate, should not be considered to the purposes of branched water-soluble polymers disclosed herein to be limited in form this conjugate purposes on.It will be understood by those skilled in the art that branched water-soluble polymers of the present invention can be used for forming various branched water-soluble polymers conjugate.
As described in part above, it is chemically conjugated that the art-recognized chemical process of covalency PEGization relies on by the reactive group on amino acid or carbohydrate.By carefully designing conjugate and reaction conditions, the conjugation strategy of chemistry mediation has been used to prepare effective conjugate.The chemically conjugated main drawback of polymkeric substance and albumen or glycoprotein is the optionally shortage of activated polymer, and this usually causes polymkeric substance affecting albumen or glycoprotein biological activity connects on site.Develop several method and solve site selectivity conjugation chemistry, but, only have developed a kind of general method being suitable for various recombinant protein.
Contrary with art-recognized method, the sugar that the invention provides the high selectivity of branched water-soluble polymers site-directed is puted together, the such as novel means of sugar-PEGization.In an exemplary embodiment of the present invention, the site-directed connection of branched water-soluble polymers has been come by the external enzymatic glycosylation of specific peptide sequences.Sugar puts together that can adopt can by material branched water-soluble polymers-glycosyl, and such as the glycosyltransferase of glycosylation site transferred to by PEG-sialic acid, and such as sialyltransferase is undertaken (" sugar-PEGization ") by enzymatic method.
Branched water-soluble polymers
In first, the invention provides the branched water-soluble polymers with following general formula:
WSP-Y-R x
Wherein WSP is water-soluble polymers.Symbol Y represents connection base, such as key, or the structure division containing acid amides, carboxylicesters, urea alkane, mercaptan, replacement or non-substituted alkyl etc.Exemplary connection base comprises: key, (CH 2) n, (CH 2) mc (O) O (CH 2) n, (CH 2) mc (O) NH (CH 2) n, (CH 2) moC (O) NH (CH 2) n, (CH 2) mo (CH 2) n, (CH 2) mnH (CH 2) n, and (CH 2) ms (CH 2) n, wherein m and n is the integer independently selected from 0-6.R xbe connected to the replacement of water-soluble polymers or non-substituted alkyl structure division; Be connected to the amino acid of water-soluble polymers or amino acid whose dimer; Or be connected to sugar or the sugar nucleotide of water-soluble polymers.WSP and R xwater-soluble copolymer component can be identical water-soluble polymers or different water-soluble polymerss.
Exemplary water soluble polymkeric substance for compound of the present invention comprises m-PEG, PEG, m-PPG, PPG, Polysialic acid, polyglutamic acid, poly aspartic acid, polylysine, polymine, polylactide, polyglycerol ester, and functionalized PEG, such as end-functionalization PEG.
In an exemplary embodiment, Y is substituted alkyl, and the invention provides the branched water-soluble polymers with following general formula:
Wherein X and Y is independently selected from OR 1, NR 2r 3, SR 4, COOR 5, CONR 6r 7, OCONR 6r 7, replace and unsubstituted alkyl, and replacement and unsubstituted aryl in member.Z 1be selected from OR 1', NR 2' R 3', SR 4', COOR 5', CONR 6' R 7', replace and non-substituted alkyl, and replace and unsubstituting aromatic yl in member.Symbol R 1, R 4and R 5represent water-soluble polymers.R 2, R 3, R 6and R 7independently selected from H, replace and non-substituted alkyl, replace and unsubstituting aromatic yl, replace and non-substituted heteroaryl, replace and unsubstituting heterocycle alkyl, reactive functional groups, and member in water-soluble polymers, prerequisite selects these groups, makes to comprise at least two water-soluble polymer structure parts according to the compound of general formula I.Symbol R 1', R 2', R 3', R 4', R 5', R 6' and R 7' represent independently selected from H, replace and non-substituted alkyl, replace and unsubstituting aromatic yl, replace and non-substituted heteroaryl, replace and unsubstituting heterocycle alkyl, reactive functional groups, the group in carrier molecule and water-soluble polymers.
In another exemplary embodiment, Z 1comprise glycosyl (saccharyl) structure division.Glycosyl structure part can be activated glycosyl structure division, such as nucleotide sugar.In addition, Z 1the amino acid whose glycosyl structure part being directly connected in peptide can be comprised, or be indirectly connected to the amino acid whose glycosyl structure part of peptide by it with being connected to amino acid whose puting together of glycosyl residue.
Following present the exemplary compounds of the present invention according to general formula I:
Wherein R 14oH or reactive functional groups.Exemplary reaction functional group is C (O) Q ', wherein selects Q ', makes C (O) Q ' be reactive functional groups.Q ' can also comprise carrier molecule (" part ").The Exemplary materials of Q ' comprises halogen, NHS, pentafluorophenyl group, HOBT, HOAt, and p-nitrophenyl.Subscript " m " and subscript " n " are independently selected from 1-20, the integer in 000.
Above-claimed cpd and other compound of the present invention can easily be prepared by such as following and so on starting raw material:
Following present the exemplary pathway obtaining compound of the present invention:
Following present another exemplary pathway obtaining compound of the present invention:
Present invention also offers the branched polymer based on amino acid or oligomeric amino acid (such as dipeptides, tripeptides, tetrapeptide).Exemplary amino acid pattern branched polymer have be selected from following among general formula:
Wherein R 11, R 11', R 12, R 12', R 13and R 13' independently selected from H, replace or non-substituted alkyl and water-soluble polymers, prerequisite selects these groups, makes above-claimed cpd comprise at least two water-soluble polymer structure parts.R 14be selected from OH, reactive functional groups, the group of sugary structure division or the one be connected in the group of carrier molecule.A is selected from NH, the one in O and S.Subscript " s " represents the integer of 1-5.A is a member be selected from NH, O and S.
Each compound described in above general formula can be used for the chemical PEGylation of other material (such as nucleic acid, peptide, sugar etc.).The method forming the conjugate between PEG (and the material containing PEG) is well known in the art usually.Such as, see Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; With people such as Feeney, MODIFICATION OF PROTEINS; Advances in ChemistrySeries, Vol.198, American Chemical Society, Washington, D.C., 1982.
In another exemplary embodiment, R 14comprise glycosyl structure part.This glycosyl structure part can be activated glycosyl structure division, such as nucleotide sugar.In addition, R 14the amino acid whose glycosyl structure part being directly connected in peptide can be comprised, or be indirectly connected to the amino acid whose glycosyl structure part of peptide by it with being connected to amino acid whose puting together of glycosyl residue.
Exemplary composition of the present invention comprises:
Such as:
Wherein " m ", " n " and " t " is independently selected from 1-20, the integer in 000; And R 14as mentioned above.
Other exemplary compounds comprises:
Such as:
Give other composition based on amino acid structure in the following table:
In the figure provided in upper table, symbol a and b represents the numerical value of 1-10 independently.Symbol m and o represents 1-10 independently, the numerical value of 000.Sign X OH, H, Q (activating group) and biological structure part, such as albumen, sugar, lipid, or Nucleotide.
In another exemplary embodiment, R 14comprise glycosyl structure part.This glycosyl structure part can be activated glycosyl structure division, such as nucleotide sugar.In addition, R 14the amino acid whose glycosyl structure part being directly connected in peptide can be comprised, or be indirectly connected to the amino acid whose glycosyl structure part of peptide by it with being connected to amino acid whose puting together of glycosyl residue.
In yet another aspect, the invention provides the branched water-soluble polymers based on sugared core (" a type core ").Technician is clear, and sugar endorses to have any structure.Exemplary sugar for this respect of the present invention comprises GlcNAc, Gal, Sia, Fuc, Glc, Gal NAc, GalNH 2, GlcNH 2deng.
Exemplary compounds of the present invention has following general formula:
Sugar-O-(L-WSP) 2
Wherein L is connection base and WSP is water-soluble polymers.
Other exemplary compounds of the present invention has following general formula:
(C 6H 10O 4)-(OC(O)-L-WSP) 2
Wherein C 6h 10o 4be a sugar core, wherein two sugared OH structure divisions are converted into OC (O)-connection base-WSP.
Another exemplary compounds of the present invention has following general formula:
Nucleotide-sugar-O-(L-WSP) 2
In another exemplary, glycosyl branched water-soluble polymers of the present invention has following general formula:
Nu-O-(C 6H 9O 3)-(OC(O)-L-WSP) 2
Wherein Nu is Nucleotide.
Another exemplary compounds (based on sialic acid core) according to this aspect of the invention has following general formula:
Wherein R 16and R 16' be selected from H, the member in ethanoyl and general formula (I):
And R 17, R 18, R 19and R 19' be independently selected from H, OH, NH 2, NHAc and according to the member in the structure division of general formula I.In general formula I, Z 2be selected from O, S, CH 2with the one in S.R 11as mentioned above, and subscript " a " represents the integer of 0-20, and prerequisite is R 16, R 16', R 17, R 18, and R 19at least two structures had according to general formula I.R 11can also be connected to the group of carrier molecule or be bonded in the key of carrier molecule.R 15be selected from H and activating group, such as, a member in phosphoric acid nucleoside acid.
In another exemplary embodiment, the connection base of general formula I has following structural formula:
In another exemplary, the connection base of general formula I has following structural formula:
Wherein Z 3be selected from NH, a member in O and S.
In an exemplary embodiment, Z 2nH.
In Still another example embodiment, the invention provides the compound with following structure:
Wherein L connects base as herein defined.
In yet another aspect, branched polymer is based on semi-lactosi or N-acetyl galactose, and it has following general formula:
Wherein R 15-R 19as mentioned above, R 15-R 19at least two be structure division according to general formula I.
In addition, R 15can comprise the amino acid whose key being connected to peptide, or be connected to the key of glycosyl structure part, this glycosyl structure part is bonded directly to the amino acid of peptide, or by put together in connection amino acid whose glycosyl residue and indirect linkage in the amino acid of peptide.
The following provide the exemplary arrangement of preparation branching sugar of the present invention core water-soluble polymers:
Following present another exemplary arrangement preparing sugared core branched water-soluble polymers of the present invention:
Single dispersing PEG
Present invention also offers mono-disperse polymer amount PEG and prepare the method for basic single dispersing colony of PEG molecule.The method comprises allows the difunctionality activated PEG also with clear and definite molecular weight of PEG molecule such as PEG200 and at least 2 equivalents with clear and definite molecular weight, such as PEG200 contact, thus produces the single dispersing sample of PEG, such as PEG 600:
G is leavings group, such as sulphonate or tresylate ester.Then the single dispersing sample of PEG600 can contact with difunctionality activated PEG 200, forms single dispersing PPEG100.Or single dispersing PEG600 can be converted into corresponding Bifunctionalized derivative, then react with the single dispersing dihydroxyl-PEG600 of at least 2 equivalents, produce single dispersing PEG1800.Repeat method of the present invention, until obtain the single dispersing PEG of required size.This synthesis can be designed, make the molecular weight differences between starting raw material and product allow to be separated by size exclusion chromatography, the material of any unreacted or partial reaction.
And as to preparing modified water-soluble polymer, the response of the demand of improving one's methods of such as PEG, the invention provides chemical activation and extends the method for polymer backbone.Single activated PEG molecules can be used for being puted together by PEG in various material, such as target structure division, treatment structure division, antitumor drug, cytotoxin, radiological agent, amino acids, sugar etc.
Therefore, in yet another aspect, the invention provides activated water-soluble polymer, the method for the especially substep assembling of PEG and analog thereof.This method provide the convenient way obtaining monofunctional and Bifunctionalized PEG molecule.
In step a, original glycol contacts with activating group (R-Y), and the hydroxyl moiety of this activating group and glycol reacts.Y is leavings group normally, and R is placed on one of hydroxyl moiety of PEG molecule.In stepb, the group that the free hydroxyl group of gained adducts is converted into sulfonic acid esters by it activates.Activation PEG material with as initial PEG (" PEG m") the identical or different polymerization degree another PEG structure division contact.In order to make and it is connected to another material, RO-PEG (n+m)optionally activate in free hydroxyl group structural portion office.
Single dispersing PEG of the present invention is easily activated by art-recognized method, and the derivative of activation can be used for forming conjugate.Or this single dispersing PEG is introduced in the branching PEG of the present invention for forming conjugate.
Water-soluble polymers
The wetting ability of the peptide selected is by such as improving containing amine, ester, hydroxyl and polyhydric molecular conjugate with polar molecule.Representative example is including, but not limited to polylysine, polymine, and polyethers, such as PEG, m-PEG, poly-(propylene glycol), m-gathers (propylene glycol), and poly-(aklylene glycol) structure division of other O-alkyl.Preferred water-soluble polymers is non-fluorescence substantially, or sends the fluorescence of minimum and be not suitable for being used in tagged fluorescent agent in analysis.And preferably use does not belong to the polymkeric substance of naturally occurring sugar usually.This preferred exception uses the naturally occurring sugar by another entity covalently bound (such as PEG, gather (propylene glycol), biomolecules, treatment structure division, diagnosis structural portion is graded) modification.In another exemplary embodiment, treat and put together in connecting arm with sugared structure division, and sugar-connecting arm box (cassette) is puted together in peptide by method of the present invention subsequently.
For activated water-soluble polymer and sugar method and chemical process and for by sugar and polymeric conjugation state in the literature in the method for various material.The method of normally used activated polymer comprises functional group's cyanogen bromide, periodate, glutaraldehyde, di-epoxide, Epicholorohydrin, divinylsulfone, carbodiimide, sulfonic acid halide, the activation of three chlorotriazines etc. (see R.F.Taylor, (1991), PROTEIN IMMOBILISATION.FUNDAMENTALS ANDAPPLICATIONS, Marcel Dekker, N.Y.; S.S.Wong, (1992), CHEMISTRYOF PROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; The people such as G.T.Hermanson, (1993), IMMOBILIZED AFFINITY LIGANDTECHNIQUES, Academic Press, the people such as N.Y., Eds.POLYMERIC DRUGS ANDDRUG DELIVERY SYSTEMS, ACS Symposium Ser ies Vol.469, AmericanChemical Society, Washington, D.C.1991).
Many water-soluble polymerss are that those skilled in the art is known, and can be used for implementing the present invention.Term water-soluble polymers comprises such as following and so on material: sugar (such as dextran, amylose starch, hyaluronic acid, poly-(sialic acid), heparan, heparin etc.; Poly-(amino acid); Nucleic acid; Synthetic polymer (such as poly-(vinylformic acid), poly-(ether), such as PEG; Peptide, albumen etc.The present invention can implement with any water-soluble polymers, and unique restriction is the site that this polymkeric substance must comprise the remainder that can connect conjugate.
Method for activated polymer can also at WO 94/17039, US patent No.5, 324, 844, WO 94/18247, WO 94/04193, US patent No.5, 219, 564, US patent No.5, 122, 614, WO 90/13540, US patent No.5, 281, 698 and WO93/15189 in find, and in order to put together activated polymer and peptide, such as blood coagulation factor VIII (WO 94/15625), oxyphorase (WO 94/09027), take oxygen molecule (US patent No.4, 412, 989), rnase and the superoxide-dismutase (people such as Veronese, App.Biochem.Biotech.11:141-45 (1985)).
Preferred water-soluble polymers is that wherein in the sample of polymkeric substance, most of polymer molecule has those of approximately identical molecular weight; These polymkeric substance are " single dispersing ".
The present invention illustrates with further reference to PEG or mono methoxy-PEG (m-PEG) conjugate.Can obtain about the functionalized of PEG and several sections of comments of puting together and monograph.Such as, see Harris, Macronol.Chem.Phys.C25:325-373 (1985); Scouten, Methods in Enzymology 135:30-65 (1987); The people such as Wong, Enzyme Microb.Technol.14:866-874 (1992); The people such as Delgado, Critical Reviews in Therapeutic Drug Carrier Systems 9:249-304 (1992); Zalipsky, Bioconjugate Chem.6:150-165 (1995); With people such as Bhadra, Pharmazie, 57:5-29 (2002).
The PEG that can be used for being formed conjugate of the present invention is linear or branching.Any molecular weight, the PEG structure division of such as 5Kd, 10Kd, 20Kd and 30Kd can be used for the present invention.
Reactive functional groups
The reactive derivatives of PEG (or other connects base) is used for one or more peptide structure division to be connected to the purposes of this connection base within the scope of the invention.The present invention does not limit by the identity of reactive PEG analogue.Many activated derivatives of PEG can be commercial, and state in the literature.If the suitable activated PEG derivative selected and need synthesis to can be used to prepare substrate used in the present invention is in the limit of power of those skilled in the art completely.See people such as Abuchowski, Cancer Biochem.Biophys., 7:175-186 (1984); The people such as Abuchowski, J.Biol.Chem., 252:3582-3586 (1977); The people such as Jackson, Anal.Biochem., 165:114-127 (1987); The people such as Koide, Biochem Biophys.Res.Commun., 111:659-667 (1983)), tresylate (people such as Nilsson, Methods Enzymol., 104:56-69 (1984); The people such as Delgado, Biotechnol.Appl.Biochem., 12:119-128 (1990)); The derivative active ester of N-hydroxysuccinimide (people such as Buckmann, Makromol.Chem., 182:1379-1384 (1981); The people such as Joppich, Makromol.Chem., 180:1381-1384 (1979); The people such as Abuchowski, Cancer Biochem.Biophys., 7:175-185 (1984); The people such as Katre, Proc.Natl.Acad.Sci.U.S.A., 84:1487-1491 (1987); The people such as Kitamura, Cancer Res., 51:4310-4315 (1991); The people such as Boccu, Z.Naturforsch., 38C:94-99 (1983), carbonic ether (people such as Zalipsky, POLY (ETHYLENE GLYCOL) CHEMISTRY:BIOTECHNICAL AND BIOMEDICAL APPLICATIONS, Harris, Ed., Plenum Press, New York, 1992, pp.347-370; The people such as Zalipsky, Biotechnol.Appl.Biochem., 15:100-114 (1992); The people such as Veronese, Appl.Biochem.Biotech., 11:141-152 (1985)), formic acid imidazate (people such as Beauchamp, Anal.Biochem., 131:25-33 (1983)); The people such as Berger, Blood, 71:1641-1647 (1988), the 4-dithiopyridines class (people such as Woghiren, Bioconjugate Chem., 4:314-318 (1993)), isocyanic ester (people such as Byun, ASAIO Journal, M649-M-653 (1992)) and epoxide (US patent No.4,806,595, license to the people such as Noishiki (1989).Other connection base is included in the urethane bond between amino and activated PEG.See people such as Veronese, Appl.Biochem.Biotechnol., 11:141-152 (1985).
Can be used for implementing reactive group of the present invention and reaction type normally in bioconjugates chemical field known those.Those that carry out under relatively mild conditions with the reaction type favourable at present that reactive sugars structure division obtains.These including, but not limited to nucleophilic substitution (such as; the reaction of amine and alcohol and acyl halide, active ester); electrophilic substitution (such as enamine reaction) and the addition on carbon-to-carbon and carbon-heteroatom multiple bond are (such as; Michael reacts, Diels-Alder addition).These and other useful reaction such as at March, ADVANCEDORGANIC CHEMISTRY, 3 rded., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; And the people such as Feeney, MODIFICATION OF PROTEINS; Advances inChemistry Series, Vol.198, American Chemical Society, Washington, D.C., state in 1982.
The useful reactive functional groups hung on the upside of sugared core or modified group including, but not limited to:
(a) carboxyl and various derivative thereof, including, but not limited to N-hydroxysuccinimide ester, N-hydroxybenzotriazole ester, carboxylic acid halides, acylimidazole, thioesters, p-nitrophenyl ester, alkyl, alkenyl, alkynyl and aromatic ester;
(b) hydroxyl, it can be converted into such as ester, ether, aldehyde etc.;
(c) haloalkyl, wherein halogen can afterwards by nucleophilic group such as amine, carboxylate anion, thiol anion, carboanion, or alkoxide ion displacement, thus cause new group to connect with covalent linkage in the functional group of halogen atom;
(d) dienophile group, it can participate in Diels-Alder reaction, such as maleimide base group;
E () aldehydes or ketones group, is made subsequent derivation such as, by forming carbonyl derivative, imines, hydrazone, semicarbazone or oximes, or is undertaken by the mechanism of such as Ge Liya addition or lithium alkylide addition and so on;
F () for reacting with amine subsequently, such as, forms the sulfonyl halide groups of sulphonamide;
(g) thiol group, it such as can be converted into disulphide or react with acyl halide;
(h) amine or sulfydryl, it such as can carry out acidylate, alkylation or oxidation;
(i) alkene, they such as can carry out cycloaddition, acidylate, Michael addition etc.; With
J () epoxide, they such as can react with amine and oxy-compound.
Can selective reaction functional group, they are not participated in, or interference assembling reactive sugars core or the necessary reaction of modified group.Or reactive functional groups can by providing protecting group and protected, in order to avoid participate in reaction.Skilled in the art will recognize that and how to protect particular functional group, make it not interfere a group reaction condition of selection.About the example of effective protecting group, such as, referring to people such as Greene, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.
Peptide conjugate
The application of compound of the present invention is illustrated by their purposes in the peptide conjugate forming branched water-soluble polymers.The focus discussed is to clearly demonstrate.Technician understands, this discussion is relevant to the method that use branched water-soluble polymers of the present invention forms various conjugate.In an exemplary embodiment, chemical reactivity branched water-soluble polymers puts together the complementary interaction group on peptide by methods known in the art or its modification.
In another exemplary, branched water-soluble polymers comprises the sugared structure division as a type core, or this branched water-soluble polymers is connected to sugar.Sugar is substrate glycosyl branched water-soluble polymers (or sugar-branched water-soluble polymers conjugate) being transferred to the amino acid of peptide or the enzyme of glycosyl residue.It will be apparent to those skilled in that, aforesaid method is not limited to implement with peptide, but is widely used in other material, such as lipid, glycolipid, other treatment structure division of sugared core.
Conjugate of the present invention is connected by the enzymatic of the sugar of branched water-soluble polymers modification and glycosylation or non-glycosylated peptide and is formed.Modified sugars directly joins glycosylation position, or directly or indirectly (such as by one or more glycosyl residue) is connected to the glycosyl residue of glycosylation position.
When inserting between peptide (or glycosyl residue) and the modified group of sugar, branched water-soluble polymers-modified sugars becomes herein so-called " glycosyl connection base ".It can be " complete " that glycosyl connects base, or it can change in branched water-soluble polymers with the connection procedure of sugar, such as oxidation and reduction amination.Use the highly selective of enzyme such as glycosyltransferase, present approach provides the peptide carrying branched water-soluble polymers at one or more specific position.Therefore, according to the present invention, modified sugars is directly connected in the selected location on peptide chain, or modified sugars is connected to the carbohydrate moiety of glycopeptide.Wherein modified sugars is bonded in glycopeptide carbohydrate and the peptide being directly bonded in the amino-acid residue of peptide backbone is also within the scope of the invention.
Different with chemo-enzymatic peptide Processing Strategies from known chemistry, the invention provides the peptide-and glycopeptide-conjugate with uniform deriving mode substantially; The enzyme adopted in the present invention generally to the particular amino acid residue of peptide or the combination of amino-acid residue selective.Conjugate of the present invention also can to prepare on a large scale.Therefore, the invention provides the practical approach that extensive preparation has the glycopeptide of preselected even derivatize mode.These methods are particularly suitable for the modification of therapeutic peptide, including, but not limited at Cell Culture Cells (such as mammalian cell, insect cell, vegetable cell, fungal cell, yeast cell or prokaryotic cell prokaryocyte) or transgenic plant or animal in incomplete glycosylated glycopeptide in process of production.
The branched water-soluble polymers conjugate of peptide is generally characterized as being the treatment transformation period having and increase, such as, due to the clearance rate lowered, or the speed absorbed by immunity or reticuloendothelial system (RES) lowered.And the antigenic determinant on the peptide composition of conjugate of the present invention shields with branched water-soluble polymers, reduce or eliminate the immune response of host to this peptide.Use the directed agents of suitable modified sugars to be connected with the selectivity of peptide to can also be used to and peptide is led to the special particular organization of specific directed agents or cell surface receptor.
The Half-life in vivo for the treatment of glycopeptide can also strengthen with the branched water-soluble polymers comprising polyoxyethylene glycol (PEG, m-PEG) and polypropylene glycol (PPG).Such as, albumen adds their molecular dimension by the chemical modification (PEGization, m-PEGization) of branching PEG and reduces their surface-and functional group-accessibility, and the latter depends on the size of the PEG being connected to albumen separately.The modification of peptide water-soluble polymers is generally acknowledged as and improves plasma half-life and proteolytic stability, and reduce promising strategy (people such as Chaffee, the J.Clin.Invest.89:1643-1651 (1992) of immunogenicity and liver picked-up; The people such as Pyatak, Res.Commun.Chem.Pathol Pharmacol.29:113-127 (1980)).The PEGization having reported interleukin-2 improves the antitumor potential (people such as Katre in its body, Proc.Natl.Acad.Sci.USA.84:1487-1491 (1987)) and the PEGization of F (ab ') 2 that obtained by monoclonal antibody A7 improve its tumor-localizing (people such as Kitamura, Biochem.Biophys.Res.Commun.28:1387-1394 (1990)).Therefore, in a further preferred embodiment, improved relative to the Half-life in vivo of non-derivative peptide by the Half-life in vivo of the peptide of method water-soluble polymers derivatize of the present invention.
The percent ranges that the increase of the peptide Half-life in vivo of conjugate of the present invention increases as this amount best represents.The lower limit of the scope that percentage increases is about 40%, about 60%, about 80%, about 100%, about 150% or about 200%.The upper limit of this scope is about 60%, about 80%, about 100%, about 150% or exceed about 250%.
In an exemplary embodiment, the intact glycosyl that the connection between peptide and choice structure part comprises between peptide and water-soluble polymers connects base.As described herein, the connection (or use of a sugar type core) of water-soluble polymers and sugared structure division provides by " modified sugars " of suitable transferring enzyme identification, and modified sugars is connected to peptide by it.When inserting between peptide and choice structure part, the sugar component of modified sugars becomes " glycosyl connection base ", such as " intact glycosyl connection base ".This glycosyl connects base and is formed by any monose or oligosaccharides, and after with water-soluble polymers modification, it is the substrate of suitable transferring enzyme.
Conjugate of the present invention corresponds to following general formula usually:
Wherein symbol a, b, c, d and s represents positive nonzero integer; And t is 0 or positive integer." reagent " is branched water-soluble polymers of the present invention.Or sugar-reagent is provided by the branched water-soluble polymers propping up type core based on sugar.This connection base can be any one of hereafter various connection base.Or this connection base can be singly-bound or " zero level connection base ".The identity of peptide is not restriction.
In an exemplary embodiment, this water-soluble polymers is PEG, m-PEG, PPG or m-PPG, and this branched water-soluble polymers is connected to peptide via complete glycosyl connection base with covalent linkage.Glycosyl connects base is connected to peptide amino-acid residue or glycosyl residue with covalent linkage.Or this glycosyl connects one or more glycosyl units that base is connected to glycopeptide.Present invention also offers wherein glycosyl and connect the conjugate that base (such as GalNAc) is connected to amino-acid residue (such as Thr or Ser).
Being connected except the conjugate of base formation by the intact glycosyl of enzymatic addition except providing, the invention provides the conjugate that it replaces mode high uniformity.Use method of the present invention, can form peptide conjugate, wherein substantially all in the colony of conjugate of the present invention modified sugars structures parts are connected to the identical amino acid of structure or glycosyl residue.Therefore, in second, the invention provides the peptide conjugate with branched water-soluble polymers structure division colony, these structure divisions connect base by glycosyl, and such as intact glycosyl connects base and is connected to peptide with covalent linkage.In preferred conjugate of the present invention, substantially each member of this colony connects via glycosyl the glycosyl residue that base key is connected to peptide, and each glycosyl residue that glycosyl connects the peptide that base connects has identical structure.
Present invention also offers the peptide conjugate with the colony being connected the branched water-soluble polymers structure division that base connects with covalent linkage by intact glycosyl.In preferred embodiments, substantially each member of the colony of branched water-soluble polymers structure division connects via complete glycosyl the amino-acid residue that base key is connected to peptide, and each amino-acid residue with connection intact glycosyl connection base thereon has same structure.
Present invention also offers and those similar conjugates above-mentioned, wherein this peptide is puted together in treatment structure division via intact glycosyl connection base further, diagnosis structure division, target structure division, toxin structure part etc.Said structure part can be small molecules separately, natural polymer (such as, polypeptide) or synthetic polymer.
In still another embodiment, the invention provides the directed agents owing to existing as the component of conjugate, the selective fixed conjugate being arranged in particular organization.In an exemplary embodiment, this directed agents is albumen.Exemplary Proteins comprises siderophilin (brain, blood pond), HS-glycoprotein (bone, brain, blood pond), antibody (brain, there is the tissue of antibody-specific antigens, blood pond), factor V-XII (damaged tissue, grumeleuse, cancer, blood pond), serum protein, such as alpha-acid glycoprotein, Pp63 glycophosphoproteins, α-fetoprotein (brain, blood pond), beta 2-glycoprotein (liver, atherosclerotic plaque, brain, blood pond), G-CSF, GM-CSF, M-CSF, with EPO (immunostimulation, cancer, blood pond, red blood corpuscle excessively produces, neuroprotective), albumin (transformation period increase), IL-2 and IFN-α.
In an exemplary embodiment, this conjugate is formed between branched water-soluble polymers and glycosylation or non-glycosylated peptide.This polymkeric substance, treatment connect base by structure division or biomolecules via intact glycosyl and put together in peptide, and this connection base inserts between peptide and modified group (such as water-soluble polymers), and are connected to the two with covalent linkage.The method comprises allows peptide contact with the mixture of the glycosyltransferase containing modified sugars and modified sugars being its substrate.This reaction is being enough to carry out under the condition forming covalent linkage between modified sugars and peptide.The sugared structure division of modified sugars is preferably from nucleotide sugar, activation sugar with not only selected non-nucleotide but also non-activated sugar.
Acceptor peptide (glycosylation or non-glycosylated) is de novo synthesis usually, or recombinant expressed in prokaryotic cell prokaryocyte (such as bacterial cell, such as E.coli) or eukaryotic cell such as Mammals, yeast, insect, fungi or vegetable cell.This peptide can be full-length proteins or fragment.And this peptide can be the peptide of wild-type or sudden change.In an exemplary embodiment, peptide comprises the sudden change one or more total glycosylation site being joined peptide sequence.
Method of the present invention is also provided for the modification of the incomplete glycosylated peptide of recombinant production.The glycopeptide of many recombinant production, by glycosylation by halves, exposes and may have undesirable performance, such as immunogenicity, the carbohydrate residue that identified by RES.Use modified sugars in the method for the invention, peptide can simultaneously further glycosylation and such as use water-soluble polymers, the derivatizes such as therapeutical agent.The sugared structure division of modified sugars can be the residue of the acceptor suitably puted together in complete glycosylated peptide, or has another glycosyl structure part of desired properties.
Give the exemplary peptides component of conjugate of the present invention in Table 1.
Table 1
Other exemplary peptides component of conjugate of the present invention comprises member's (such as antibody of immunoglobulin class, MHC molecule, φt cell receptor etc.), iuntercellular acceptor (such as integrin, the acceptor etc. of hormone or somatomedin) lectin, and cytokine (such as interleukin).Other example especially comprises tissue plasminogen activator (t-PA), feritin, thrombin is factor Ⅴ-XII such as, bombesin, zymoplasm, hemopoieticgrowth factor, G CFS, virus antigen, complement proteins, alpha1-antitrypsin, erythropoietin, CD62P glycopeptide part-1 (PSGL-1), granulocyte-macrophage G CFS, Antithrombin III, interleukin, Interferon, rabbit, a-protein and C, Fibrinogen, Trastuzumab (herceptin), Leptin (leptin), Glycosylase, HS-glycoprotein, serum protein (such as, alpha-acid glycoprotein, Pp63 glycophosphoproteins, α-fetoprotein), beta 2-glycoprotein.This polypeptide list is exemplary, is not exclusiveness.The peptide composition of conjugate can also comprise fusion rotein and chimeric protein, including, but not limited to the chimeric protein contained by the fragment such as structure division that FAb (Fc district) is derivative of immunoglobulin (Ig) such as IgG or immunoglobulin (Ig).In addition, giving in annex 1 can by the exemplary peptides of method modification of the present invention.Exemplary peptides provided herein is used to provide the selection that can be used for implementing peptide of the present invention; Therefore, they are not restrictive.Technician is clear, and the present invention can use and the substantially all peptides from any source can be used to implement.
The peptide composition of conjugate of the present invention can be synthesis or wild type peptide, or they can be the peptides of sudden change, and the latter's methods known in the art, such as directed mutagenesis is produced.The glycosylation of peptide normally N-connect or O-connects.Exemplary N-connection is the connection of the side chain of modified sugars and asparagine residue.Tripeptide sequence asparagine-X-serine is the recognition sequence that carbohydrate moiety is connected with the enzyme process of asparagine side chain with asparagine-X-threonine (wherein X is any amino acid except proline(Pro)).Therefore, the existence of any one of these tripeptide sequences in polypeptide creates potential glycosylation site.The glycosylation that O-connects refers to a sugar (such as GalNAc; semi-lactosi; seminose; GlcNAc; glucose, Fucose, wood sugar) and hydroxy-amino-acid; the connection of the hydroxyl side chains of preferred Serine or Threonine, although can also use 5-oxyproline or 5-oxylysine.
And except peptide, the component of the conjugate puted together with branched water-soluble polymers of the present invention can be the biological structure (such as glycolipid, lipid, sphingol, ceramide, whole cell etc.) except peptide.
Glycosylation site is joined peptide or other structure conveniently by change aminoacid sequence, it to be made to contain one or more glycosylation site and come.This interpolation can be undertaken by the full chemosynthesis of sudden change or peptide.Peptide amino acid sequence, preferably by the change under DNA level, particularly by the DNA mutation making this peptide of coding with preselected base, makes to produce and is translated as amino acid needed codon to change.DNA mutation preferably uses methods known in the art to carry out.
In an exemplary embodiment, glycosylation site is added by reorganization (shuffling) polynucleotide.The polynucleotide of the peptide of encodes candidate can reorganize operation to regulate with DNA.DNA reorganization is the method for recurrence restructuring and sudden change, by random split coil method correlation gene storehouse, re-assemblies fragment to carry out subsequently by polymerase chain reaction class methods.Such as, see Stemmer, Proc.Natl.Acad.Sci.USA 91:10747-10751 (1994); Stemmer, Nature370:389-391 (1994); With U.S. patent Nos.5,605,793,5,837,458,5,830,721 and 5,811,238.
Conjugate of the present invention can also comprise and added or removed one or more selection glycosyl residue, after this modified sugars is puted together the peptide of at least one of the selection glycosyl residue in peptide.Such as, when modified sugars to be puted together the selection glycosyl residue in not being present on peptide or do not exist with aequum by hope, this embodiment is useful.Therefore, before modified sugars is coupled to peptide, by enzymatic coupling or chemical coupling, selection glycosyl residue is puted together in peptide.In another embodiment, the glycosylation patterns of glycopeptide before puting together modified sugars by removing carbohydrate residue to change from glycopeptide.Such as, see WO 98/31826.
Any carbohydrate moiety chemical method that increase or removing are present on glycopeptide or enzyme process have come.Chemical deglycosylation has come preferably by polypeptide variants contact compound trifluoromethanesulfonic acid or equivalent compound.This process result in the cracking of great majority except connecting sugar (N-acetyl glucosamine or GalNAc) or all sugar, but keeps peptide complete.The people such as Hakimuddin, the people such as Arch.Biochem.Biophys.259:52 (1987) and Edge, Anal.Biochem.118:131 (1981) describes Chemical deglycosylation.The enzymatic lysis of the carbohydrate moiety on polypeptide variants can by use as by people such as Thotakura, and the various endoglycosidase described in Meth.Enzymol.138:350 (1987) and exoglycosidase have come.
The chemical addition of glycosyl structure part is undertaken by any method of technically generally acknowledging.The enzymatic addition of glycosyl structure part preferably uses the modification of method as herein described, replaces the modified sugars used in the present invention to carry out by natural glycosyl units.Other method adding saccharogenesis structure division is disclosed in US patent No.5,876,980,6,030,815,5,728,554 and 5,922, in 577.
For selecting the exemplary link position of glycosyl residue including, but not limited to (a) N-and O-glycosylated conserved site; B () belongs to the terminal saccharide structure division of the acceptor of glycosyltransferase; (c) arginine, l-asparagine and Histidine; (d) free carboxy; Those of (e) free sulfhydryl groups such as halfcystine; (f) free hydroxyl group, such as Serine, Threonine, or those of oxyproline; (g) aromatic moieties, such as phenylalanine, tyrosine, those of tryptophane; Or the amido of (h) glutamine.Be described in WO 87/05330 disclosed in 11 days September in 1987 and Aplin and Wriston, CRC, CRIT.REV.BIOCHEM., pp.259-306 (1981) for illustrative methods of the present invention.
In one embodiment, the invention provides the method connecting two or more peptide by connecting base.This connection base has any effective structure, can be selected from straight chain and branched structure.Preferably, each end being connected to the connection base of peptide comprises modified sugars (that is, nascent complete glycosyl connects base).
In illustrative methods of the present invention, via the syndeton part comprising branched water-soluble polymers connection base, two peptides are connected together.This constructor is combined in the general formula shown in above figure.As described herein, structure of the present invention comprises two complete glycosyls connection base (i.e. s+t=1).It is for purposes of clarity that the PEG that concentrated description comprises two glycosyls connects base, should not be considered to limit the identity for the connecting arm of this embodiment of the present invention.
Modified sugars
Modification glycosyl donating substances (" modified sugars ") is preferential from modified sugars Nucleotide, and activation modification sugar and belonging to not only was selected in non-nucleotide but also the modified sugars of non-activated simple sugars.Any desirable carbohydrate structure can be incorporated in conjugate of the present invention.Usually, this structure is monose, but the invention is not restricted to use modification monose; Oligosaccharides and polypeptide are also useful.
Modified group by enzyme mode, chemical mode or their combination and be connected to sugared structure division, thus produce modified sugars.Sugar connects modified structure part in permission, but still any position that sugar can be made to be used from the effect of the substrate of enzyme modified sugars being incorporated into peptide replaces.In a preferred embodiment, when sialic acid is sugar, sialic acid is by upper or normally in sialic acid, 5 of acetylizad amine structure part to replace by modified group at 9 of pyruvoyl (pyruvyl) side chain.
In certain embodiments of the invention, modified sugars Nucleotide is utilized modified sugars to be added on peptide.The exemplary sugar nucleotide adopted in the present invention with its modified form comprises one, two or triphosphopyridine nucleotide or their analogue.In preferred embodiments, modified sugars Nucleotide is selected from UDP-glucosides, CMP-glucosides, or GDP-glucosides.Also more preferably, modified sugars Nucleotide is selected from UDP-semi-lactosi, UDP-GalN, UDPG, UDP-glycosamine, GDP-seminose, GDP-Fucose, cmp sialic acid, or CMP-NeuAc.The N-acetyl amine derivative also method used in the present invention of sugar nucleotide.
Present invention also offers use modified sugars, such as modification semi-lactosi, modification Fucose, the method for modification GalNAc and modification sialic acid synthesis modification peptide.When adopting modification sialic acid, sialyltransferase or trans-sialidase (only for the sialic acid that α 2,3-connects) can use in these methods.
In other embodiments, modified sugars is activation sugar.Activation modification sugar used in the present invention normally changes into by synthesis the glucosides comprising Activated leaving groups.Term as used herein " Activated leaving groups " refers to those structure divisions replaced in the nucleophilic substitution reaction easily regulated at enzyme.Be known in the art many activation sugar.Such as, see people such as Vocadlo, In CARBOHYDRATE CHEMISTRY AND BIOLOGY, the people such as Vol.2, Ernst, Ed., Wiley-VCH Verlag:Weinheim, Germany, 2000; The people such as Kodama, Tetrahedron Lett.34:6419 (1993); The people such as Lougheed, J.Biol.Chem.274:37717 (1999).
The example of activating group (leavings group) comprises fluorine, chlorine, bromine, tosylate, methanesulfonates, triflate etc.That significantly steric hindrance glucosides enzyme process transfers to those on acceptor for preferred Activated leaving groups of the present invention.Therefore, the preferred embodiment of activation glycosides derivatives comprises glycosyl fluorochemical and glycosyl methanesulfonates, and wherein glycosyl fluorochemical is particularly preferred.In glycosyl fluorochemical, fluoridize α-semi-lactosi, fluoridize α-seminose; fluoridize α-glucose, fluoridize α-Fucose, fluoridize α-wood sugar; fluoridize α-sialic acid, fluoridize α-N-acetyl glucosamine, fluoridize α-GalNAc; fluoridize beta galactose, fluoridize β-seminose, fluoridize β-glucose; fluoridize β-Fucose; fluoridize β-wood sugar, fluoridize β-sialic acid, it is most preferred for fluoridizing β-N-acetyl glucosamine and fluoridizing β-GalNAc.
For example, can by first by sugared acetylize with then prepare glycosyl fluorochemical by HF/ pyridine process cause free sugar.This generates the anomer of the most stable protection of thermodynamics (acetylize) glycosyl fluorochemical (that is, alpha-glycosyl fluorochemical).If need unstable anomer (that is, β-glycosyl fluorochemical), it can by being converted into different head bromide with HBr/HOAc or HCl by full acetylated sugar or prepared by muriate.Acetylated glycosyl fluorochemical can be protected by reacting to make a return journey in methyl alcohol (such as NaOMe/MeOH) with gentle (catalysis) alkali.In addition, many glycosyl fluorochemicals can be commercial.
Other activated glycosyl derivative can use usual way well known by persons skilled in the art to prepare.Such as, methylsulfonic acid glycosyl ester can by the sugar by the complete Benzylation hemiacetal form of methylsulfonyl chloride process, and subsequent catalytic hydrogenation is to remove benzyl to prepare.
In another exemplary embodiment, modified sugars is the oligosaccharides with feeler structure.In a preferred embodiment, one or more ends of feeler carry modified structure part.When more than one modified structure part is connected to the oligosaccharides with feeler structure, this oligosaccharides can be used for " amplification " modified structure part; The modified group of multiple copy is connected to this peptide by each oligosaccharide units puted together in peptide.General structure as the of the present invention typical conjugate as shown in upper figure includes by the multivalence material adopting feeler structure to prepare conjugate of the present invention acquisition.The sugared structure of many feelers is well known in the art, and the inventive method is implemented with them with can not being with restriction.
Generally, sugared structure division and modified group connect together by using reactive group, and this reactive group is converted into new organo-functional group or unreacted material by method of attachment usually.Sugar reactive functional groups is positioned on any position of sugared structure division.
In the following discussion, the many object lessons that can be used for implementing modified sugars of the present invention are illustrated.In an exemplary embodiment, the sialic acid sugared core connecting modified group.It is to clearly demonstrate that discussion concentrates on sialic acid derivative, should not be considered to the restriction of scope of the present invention.It will be obvious to those skilled in the art that other sugared structure division multiple can carry out activating and derivatize according to the described as an example similar mode of use sialic acid.Such as, many methods can be used for modification semi-lactosi, glucose, and GalNAc and Fucose provide several sugared substrate, and they easily carry out modification by art-recognized method.Such as, see people such as Elhalabi, Curr.Med.Chem.6:93 (1999); With people such as Schafer, J.Org.Chem.65:24 (2000).
In an exemplary embodiment, at prokaryotic cell prokaryocyte (such as E.coli.) by the peptide of method modification of the present invention, comprise the eukaryotic cell of yeast and mammalian cell (such as, Chinese hamster ovary celI), or in transgenic animal, producing the glycopeptide of the oligonucleotide chain also therefore containing N-and/or O-connection, they are by not exclusively sialylated.Lack sialic acid but the oligonucleotide chain of glycopeptide containing terminal galactose residues can carry out Glycopegylated, sugared PPGization or with the modification of modification sialic acid.
In scheme 2, the aminoglycoside 1 active ester process of protected amino acid (such as glycine) derivative, is converted into corresponding protected amino acid amide adduct by glucosamine residue.The zymohexase process of this adducts, forms alpha-hydroxy carboxylic acid compounds salt 2.By the effect of CMP-SA synthetic enzyme, compound 2 is converted into corresponding CMP derivative, this CMP derivative of subsequent catalytic hydrogenation, forms compound 3.By allowing compound 3 and activation (m-) PEG or (m-) PPG derivative (such as PEG-C (O) NHS, PPG-C (O) NHS) reaction, form 4 or 5 respectively, the amine introduced via formation glycine adduct is used as the site that PEG or PPG connects.
Scheme 2
Wherein X-BWSP is activation branched water-soluble polymers of the present invention, and BWSP is branched water-soluble polymers.
Table 2 gives the representative example of the sugared phosplate with PEG or PPG structure division derivatize.Some compound of table 2 is prepared by the method for scheme 4.Other derivative is prepared by art-recognized method.Such as, see people such as Keppler, Glycobiology 11:11R (2001); With people such as Charter, Glycobiology 10:1049 (2000).Reactive PEG and the PPG analogue of other amine can be commercial, or prepared by the method that they can be convenient to obtain by those skilled in the art.
Table 2
Wherein R is branched water-soluble polymers of the present invention.
Can be used for implementing modified sugars phosphoric acid ester of the present invention in other position and above-mentioned those to be substituted.Following present sialic preferred replacement at present:
Wherein X connects base, is preferentially selected from-O-,-N (H)-and ,-S, CH 2-and-N (R) 2, wherein each R is independently selected from R 1-R 5in a member.Symbol Y, Z, A and B represent the group in the group be selected from described in the above identity for X separately.X, Y, Z, A and B can select independently of one another, and therefore, they can be identical or different.Symbol R 1, R 2, R 3, R 4and R 5represent H, or branched water-soluble polymers.In addition, these symbols represent the connection base being bonded in branched water-soluble polymers.
crosslinked group
Preparation for the modified sugars of method of the present invention comprises and modified group is connected to saccharide residue and forms stable adducts, and the latter is the substrate of glycosyltransferase.Sugar and modified group can carry out coupling by zero level or senior linking agent.Can be used for modified group to be connected to the exemplary difunctional compound of carbohydrate moiety including, but not limited to Bifunctionalized PEG, polymeric amide, polyethers, polyester etc.Known in the literature for carbohydrate being connected to the general method of other molecule.Such as, see people such as Lee, Biochemistry 28:1856 (1989); The people such as Bhatia, Anal.Biochem.178:408 (1989); The people such as Janda, the people such as J.Am.Chem.Soc.112:8886 (1990) and Bednarski, WO92/18135.In the following discussion, the reactive group on the sugared structure division of newborn modified sugars carries out favourable process.Concentrated discussion is to clearly demonstrate.It will be apparent to those skilled in that, this discussion is relevant to the reactive group in modified group.
Exemplary policy comprises use heterobifunctional agents SPDP, and (protection sulfydryl is incorporated into sugar and above and then sulfydryl is gone protection by n-succimide base-3-(2-pyridyl dithio) propionic ester, forms disulfide linkage with another sulfydryl in modified group.
If SPDP deleteriously have impact on the ability that modified sugars is used as glycosyltransferase substrate, one of other linking agent a series of such as 2-imino-sulphur ring (2-iminothiolane) or N-succimide base S-acetylthioacetate (SATA) are used for forming disulfide linkage.2-imino-sulphur ring and primary amine reaction, be incorporated into unprotected sulfydryl in the molecule containing amine immediately.SATA also with primary amine reaction, but introduce the sulfydryl of protection, it used azanol to carry out deacetylation afterwards, formed free sulfhydryl groups.In all cases, the sulfydryl introduced freely with other sulfydryl or protect sulfydryl such as SPDP to react, the disulfide linkage needed for formation.
Above-mentioned strategy is the example for connection base of the present invention, is not restrictive.Can obtain other linking agent, they can be used for Different Strategies modified group and peptide are cross-linked.Such as, (((S-(2-thiopyridine base) sulfydryl-propionyl hydrazine) is reacted with the carbohydrate moiety be oxidized by the periodate process of gentleness in advance TPCH, therefore between the hydrazides part and the aldehyde of periodate generation of linking agent, defines hydrazone key for S-(2-thiopyridine base)-Cys hydrazides and TPMPH.The sulfydryl that 2-pyridyl thioketones is protected is incorporated on sugar by TPCH and TPMPH, and it can go protection with DTT, then for puting together, is such as formed in the disulfide linkage between component.
If find that disulfide linkage is unsuitable for producing stable modified sugars, other linking agent of the more stable key introduced between component can be used.Heterobifunctional agents GMBS (N-γ-maleimidobutyryloxy) succimide) and SMCC (succimide base 4-(N-maleimidomethyl) hexanaphthene) and primary amine reaction, therefore maleimide base group is incorporated in this component.Dimaleoyl imino can react with the sulfydryl in another component subsequently, and the latter can be introduced by aforementioned crosslinking agent, because which form the stable thioether bond between component.If the steric hindrance between component disturbs the ability that the activity of any one component or modified sugars are used as glycosyltransferase substrate, linking agent can be used, it introduces long spacer arm and includes the derivative (that is, SPDP) of some of aforementioned crosslinking agent between component.Therefore, there is spendable a large amount of linking agent be applicable to; They are produced to best peptide conjugate and modified sugars the effect had according to it separately and select.
Plurality of reagents can be used to component with intramolecular chemical linking agent modified modified sugar (about the commentary of linking agent and cross-linking process step, see Wold, F., Meth.Enzymol.25:623-651,1972; Weetall, H.H., and Cooney, D.A., In:ENZYMES ASDRUGS. (Holcenberg, and Roberts, eds.) pp.395-442, Wiley, NewYork, 1981; Ji, T.H., Meth.Enzymol.91:580-609,1983; The people such as Mattson, Mol.Biol.Rep.17:167-183,1993, they are all incorporated herein for reference).Preferred linking agent is by various distance of zero mark, with difunctionality and heterobifunctional agents's acquisition.Zero-length crosslinking reagents comprises directly puting together of two intrinsic chemical groups, does not introduce foreign material.The reagent that catalyses disulfide bond is formed belongs to this kind of.Another example brings out carboxyl and primary amino condensation, and to form the reagent of amido linkage, such as carbodiimide, Vinyl chloroformate, (2-ethyl-5-phenyl is different for Woodward reagent K azoles father-in-law-3 '-sulfonate), and carbonyl dimidazoles.Except these chemical reagent, enzyme-transglutaminase (glutamyl-peptide gamma-glutamyl based transferase; EC 2.3.2.13) can zero-length crosslinking reagents be used as.This enzyme catalysis at the carboxamide groups place of protein bound glutaminyl residues usually with the acyl group shift reaction as the primary amino of substrate.Preferably contain two identical or two different loci respectively with Heterobifunctional reagent, they can have reactivity to amino, sulfydryl, guanidine radicals, indoles or nonspecific group.
I. the preferred specific site in linking agent
1, amino-reactive group
In a preferred embodiment, the site on linking agent is amino-reactive group.The useful limiting examples of amino-reactive group comprises N-hydroxysuccinimide (NHS) ester, imino esters, isocyanic ester, acyl halide, aromatic yl azide, p-nitrophenyl ester, aldehyde, and SULPHURYL CHLORIDE.
NHS ester primary (comprising aromatics) amino that is preferential and modified sugars component reacts.The imidazole group of Histidine is known to primary amine competing reaction, but reaction product is unstable and easily hydrolysis.This reaction relates to the nucleophillic attack of amine on the acidic carboxypolymer of NHS, forms acid amides, release N-hydroxysuccinimide.Therefore, the positive charge of starting amino loses.
Imino esters is the most special acylating agent for reacting with the amido of modified sugars component.Under the pH of 7-10, imino esters only with primary amine reaction.Primary amine nucleophillic attack imido-ester, forms the intermediate resolving into amidine at a high ph or be decomposed into new imido-ester at a low ph.This imidate can with another primary amine reaction, therefore make two amino be cross-linked, that is, the situation of the monofunctional imido-ester of the difunctional reactant of presumption.The amidine that alkalescence is better than initial amine with the primary product of primary amine reaction.Therefore the positive charge of starting amino is retained.
Isocyanic ester (and lsothiocyanates) and the primary amine reaction of modified sugars component, form stable key.They and the reaction of sulfydryl, imidazoles and tyrosyl obtain relative unstable product.
Acyl azide can also be used as amino specific reagent, and wherein the nucleophilic amine of affinity component is under weak basic condition, such as, attacks acidic carboxypolymer under pH8.5.
Amino and the tyrosine phenolic groups of the preferential and modified sugars component of fluoro-2, the 4-dinitrobenzenes of aryl halide such as 1,5-bis-are reacted, and react with sulfydryl and imidazole group.
The p-nitrophenyl ester of monocarboxylic acid and dicarboxylic acid is also useful amino-reactive group.Although this reagent specificity is not very high, α-and omega-amino-seem to react the most rapidly.
The primary amine reaction of aldehyde such as glutaraldehyde and modified sugars.Although define unstable Schiff when the amino reaction with aldehyde, glutaraldehyde can by stable this modified sugars cross-linking modified.At pH6-8, under the pH of typical cross linking conditions, cyclic polymer experienced by dehydration, forms α, beta-unsaturated aldehyde polymkeric substance.But Schiff is stable, when puting together in another double bond.The Resonant Interaction of two double bonds prevents the hydrolysis of Schiff key.In addition, the amine under high local concentrations can attack olefinic double bond, thus forms stable Michael adduct.
Each site of aromatics SULPHURYL CHLORIDE and modified sugars component is reacted, but is most important with the reaction of amino, defines stable sulphonamide key.
2, sulfydryl reactive group
In another preferred embodiment, this site is sulfydryl reactive group.Useful, the limiting examples of sulfydryl reactive group comprise maleimide, alkylogen, Pyridine disulfide, and thiophthalimide.
Maleimide sulfydryl that is preferential and modified sugars component reacts, thus forms stable thioether bond.They also react with the primary amino of much slow speed and Histidine and imidazole group.But pH 7 times, maleimide base group can be considered to sulfydryl specific group, because under this pH, the speed of reaction of simple mercaptan is higher than the speed of corresponding amine 1000 times.
Alkylogen and sulfydryl, sulfide, imidazoles and amino react.But under neutrality to weakly alkaline pH, the main and sulfydryl of alkylogen reacts, and forms stable thioether bond.Under higher pH, be preferential with the reaction of amino.
Pyridine disulfide reacts with free sulfhydryl groups via disulfide exchange, obtains mixed disulphide.As a result, Pyridine disulfide is the sulfydryl reactive group that specificity is the highest.
Thiophthalimide and free sulfhydryl groups react, and form disulphide.
3, carboxyl-reactive residue
In another embodiment, the carbodiimide of water soluble and organic solvent is used as carboxyl-reactive reagent.These compounds and free carboxy react, and form pseudo-urea, then it can be coupled to obtainable amine, define amido linkage, it is taught that and how to use Carbodiimide-Modified carboxyl (people such as Yamada, Biochemistry 20:4836-4842,1981).
Ii. the preferred non-specific sites in cross-linking reagent
Except the reactive structure division of locus specificity, the present invention intends to use nonspecific reaction group that sugar is connected to modified group.
The non-specific linking agent of example comprises can the group of photoactivation, being complete inertia in the dark, being converted into reactive materials when absorbing the photon of suitable energy.In a preferred embodiment, can the group of photoactivation be selected from heat or photodissociation trinitride time the precursor of nitrene class that produces.The reactivity of electron deficiency nitrene class is high, can with the various chemical bonding reactions comprising N-H, O-H, C-H and C=C.Although can use three class trinitride (aryl, alkyl and acyl derivative), aromatic yl azide is preferred at present.Aromatic yl azide when photodissociation with the reactivity of N-H and O-H than good with the reactivity of C-H.Electron deficiency aryl nitrene class widening of the ring rapidly, forms dehydrogenation azepine it tends to react with nucleophile, and non-formation C-H inserts product.The reactivity of aromatic yl azide can improve by providing in ring electron-withdrawing substituent such as nitro or hydroxyl.The maximum absorption of aromatic yl azide is pushed to longer wavelength by this type of substituting group.Unsubstituted aromatic yl azide has the maximum absorption within the scope of 260-280nm, and hydroxyl and nitroaryl trinitride absorb a large amount of light more than 305nm.Therefore, hydroxyl and nitroaryl trinitride are most preferred, because compared with unsubstituted aromatic yl azide, they allow to use not too harmful photolytic conditions to come for affine component.
In a further preferred embodiment, fluorinated aryl azides compound can be selected from by photoactivation group.The photolytic product of fluorinated aryl azides compound is aryl nitrene class, and they all carry out the characteristic reactions of this group, comprises c h bond and inserts, and has high efficiency people such as (, J.Org.Chem.55:3640-3647,1990) Keana.
In another embodiment, the group of photoactivation benzophenone residue can be selected from.Benzophenone reagent generally obtains higher cross linking yield than aromatic yl azide reagent.
In another embodiment, the group of photoactivation can be selected from diazonium compound, it forms electron deficiency carbene when photodissociation.These carbene classes experience various reaction, comprise and are inserted in c h bond, add in double bond (comprising aromatic systems), and hydrogen attracts and is coordinated in nucleophilic center, obtain carbon ion.
In still another embodiment, diazonium pyruvate can be selected from by photoactivation group.Such as, the p-nitrophenyl ester of diazonium pyruvic acid p-nitrophenyl ester reacts in aliphatic amine, and obtain diazonium pyruvic acid acid amides, the latter experiences ultraviolet photodissociation, forms aldehyde.The affine component of the diazonium pyruvate modification of photodissociation will be reacted as formaldehyde or glutaraldehyde, be formed crosslinked.
Iii. same bi-functional reagents
1, with the same bifunctional cross-linker of primary amine reaction
Synthesis, the performance and application of amine reactant cross-linker describe in the literature in a large number (for the commentary of cross-linking process step and reagent, see above).Much reagent can obtain (such as, PierceChemical Company, Rockford, I11.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR.).
Preferred limiting examples with difunctionality NHS ester comprises pentanedioic acid two succimide ester (DSG), suberic acid two succimide ester (DSS), suberic acid two (sulfo group succimide ester) (BS), tartrate two succimide ester (DST), tartrate two (sulfo group succimide) ester (sulfo group-DST), two-2-(succimide oxygen base carbonyl oxygen base) ethyl sulfone (BSOCOES), two-2-(sulfo group succimide oxygen base carbonyl oxygen base) ethyl sulfone (sulfo group-BSOCOES), ethylene glycol bis (succimide base succinate) (EGS), ethylene glycol bis (sulfo group succimide base succinate) (sulfo group-EGS), dithio two (propionic acid succimide ester) (DSP), and dithio two (propionic acid sulfo group succimide base ester) (sulfo group-DSP).Preferred with difunctionality imino esters, limiting examples comprises malonyl-imido acid dimethyl ester (DMM), succinyl imido acid dimethyl ester (DMSC), hexanedioyl imido acid dimethyl ester (DMA), pimeloyl imido acid dimethyl ester (DMP), suberoyl imido acid dimethyl ester (DMS), 3, 3 '-oxygen dipropyl imide dimethyl phthalate (DODP), 3, 3 '-(methylenedioxy) dipropyl imide dimethyl phthalate (DMDP), 3, 3 '-(dimethylene dioxy base) dipropyl imide dimethyl phthalate (DDDP), 3, 3 '-(tetramethylene dioxy base)-dipropyl imide dimethyl phthalate (DTDP), and 3, the two propionyl imido acid dimethyl ester (DTBP) of 3 '-dithio.
Preferred, limiting examples with Bifunctional phenyliso thiocyanic ester comprise: to phenylene diisothio-cyanate (DITC), and 4,4 '-two isothiocyano-2,2 '-disulfonic acid stibene (DIDS).
Preferred limiting examples with bifunctional isocyanate comprises Xylene Diisocyanate, Toluene-2,4-diisocyanate, 4-vulcabond, Toluene-2,4-diisocyanate-isocyanic ester-4-lsothiocyanates, 3-methoxyl group ditan-4,4 '-vulcabond, 2,2 '-dicarboxyl-4,4 '-azobenzene vulcabond, and hexamethylene diisocyanate.
Preferred, limiting examples with difunctionality aryl halide comprise fluoro-2, the 4-dinitrobenzenes (DFDNB) of 1,5-bis-, and 4,4 '-two fluoro-3,3 '-dinitrophenyl sulfone.
Preferred, limiting examples with difunctional aliphatic's aldehyde reagent comprise oxalic dialdehyde, mda and glutaraldehyde.
Preferred, limiting examples with difunctionality acylating agent comprise the nitrophenyl ester of dicarboxylic acid.
Preferred, limiting examples with difunctional aromatic's SULPHURYL CHLORIDE comprise phenol-2,4-disulfonic acid chloride, and naphthyl alcohol-2,4-disulfonic acid chloride.
Other amino-reactive comprises tetrahydroxybutane double manganese ester with preferred, the limiting examples of bi-functional reagents, and it can react with amine, obtains double carbamate.
2. the same bifunctional cross-linker reacted with free sulfhydryl groups
The synthesis of this type of reagent, performance and application describe in the literature (about the commentary of cross-linking process step and reagent, see above).Much reagent can commercial (such as, Pierce ChemicalCompany, Rockford, I11.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR.).
Preferred, limiting examples with bifunctional maleimides comprise dimaleoyl imino hexane (BMH), N, N '-(1,3-phenylene) bismaleimides, N, N '-(1,2-phenylene) bismaleimides, azobenzene dimaleimide, and two (N-maleimidomethyl) ether.
Preferred, limiting examples with difunctionality Pyridine disulfide comprise Isosorbide-5-Nitrae-two-3 '-(2 '-pyridyldithiol) propionamido-butane (DPDPB).
Preferred, limiting examples with difunctional alkyl halide comprise 2,2 '-dicarboxyl-4,4 '-diiodo-acetamido nitrogen benzide; α; the iodo-p-Xylol sulfonic acid of α '-two, the bromo-p-Xylol sulfonic acid of α, α '-two; N; N '-bis-(b-bromotrifluoromethane) benzyl amine, N, N '-two (acetyl bromide) phenyl hydrazine; with 1,2-bis-(acetyl bromide) amino-3-phenyl-propane.
3. can the linking agent of photoactivation with difunctionality
Synthesis, the performance and application of this type of reagent describe in the literature in a large number (for the commentary of cross-linking process step and reagent, see above).Some of these reagent can commercial (such as, PierceChemical Company, Rockford, I11.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR).
Preferred, the limiting examples of linking agent of photoactivation two-β-(4-azido-salicyl amide group) ethyl disulfide (BASED) can be comprised with difunctionality, two-N-(2-nitro-4-azido-phenyl)-cystamine-S, S-dioxide (DNCO), with 4, the two phenyl azide of 4 '-dithio.
Iv. Heterobifunctional reagent
1, there is the amino-reactive Heterobifunctional reagent of Pyridine disulfide based moiety
Synthesis, the performance and application of this type of reagent describe in the literature in a large number (for the commentary of cross-linking process step and reagent, see above).The many of these reagent can commercial (such as, PierceChemical Company, Rockford, I11.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, I nc., Eugene, OR).
There is the preferred of the Heterobifunctional reagent of Pyridine disulfide structure division and amino-reactive NHS ester, limiting examples comprises N-succimide base-3-(2-pyridyldithiol) propionic ester (SPDP), succimide base 6-3-(2-pyridyidithio) propionamido-capronate (LC-SPDP), sulfo group succimide base 6-3-(2-pyridyidithio) propionamido-capronate (sulfo group-LCSPDP), 4-succimide base oxygen base carbonyl-Alpha-Methyl-α-(2-pyridyidithio) toluene (SMPT), with sulfo group succimide base 6-Alpha-Methyl-α-(2-pyridyidithio) toluoyl amido capronate (sulfo group-LC-SMPT).
2, there is the amino-reactive Heterobifunctional reagent of maleimide structure part
Synthesis, the performance and application of this type of reagent describe in the literature in a large number.There is the preferred of the Heterobifunctional reagent of maleimide structure part and amino-reactive NHS ester, limiting examples comprises maleimide guanidine-acetic acid succimide ester (AMAS), 3-maleimidoproprionic acid succimide ester (BMPS), N-γ-maleimidobutyryloxy succimide ester (GMBS), N-γ-maleimidobutyryloxy sulfo group succimide ester (sulfo group-GMBS), succimide base 6-maleimidocaproic acid ester (EMCS), succimide base 3-maleimidobenzoate (SMB), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo group-MBS), succimide base 4-(N-maleimidomethyl)-hexanaphthene-1-carboxylicesters (SMCC), sulfo group succimide base 4-(N-maleimidomethyl)-hexanaphthene-1-carboxylicesters (sulfo group-SMCC), succimide base 4-(to maleimidophenyl) butyric ester (SMPB), with sulfo group succimide base 4-(to maleimidophenyl) butyric ester (sulfo group-SMPB).
3, there is the amino-reactive Heterobifunctional reagent of alkylogen structure division
Synthesis, the performance and application of this type of reagent describe in the literature in a large number.There is the preferred of the Heterobifunctional reagent of alkylogen structure division and amino-reactive NHS ester, limiting examples comprises N-succimide base-(4-iodoacetyl) Aminobenzoate (SIAB), sulfo group succimide base-(4-iodoacetyl) Aminobenzoate (sulfo group-SIAB), succimide base-6-(iodoacetyl) hexosamine ester (SIAX), succimide base-6-(6-((iodoacetyl)-amino) caproyl amino) capronate (SIAXX), succimide base-6-(((4-(iodoacetyl)-amino)-methyl)-hexanaphthene-1-carbonyl) hexosamine ester (SIACX), and succimide base-4 (iodoacetyl)-amino) methylcyclohexane-1-carboxylicesters (SIAC).
The preferred embodiment with the Heterobifunctional reagent of amino-reactive NHS ester and alkyl dihalide structure division is N-hydroxysuccinimide base 2,3-dibromo-propionic acid ester (SDBP).Intramolecular crosslinking is incorporated into affine component by puting together its amido by SDBP.The reactivity of dibromo propionyl structure division and primary amine groups is by controlling reaction temperature people such as (, Protein Chem.7:581-592 (1988)) McKenzie.
Preferred, the limiting examples with the Heterobifunctional reagent of alkylogen structure division and amino-reactive p-nitrophenyl ester structure part comprise iodoacetic acid p-nitrophenyl ester (NPIA).
Other linking agent is known to the person skilled in the art.Such as, see people such as Pomato, U.S. patent No.5,965,106.Selecting suitable linking agent to be used for application-specific is in those skilled in the art's limit of power.
V. the connection base of cleavable
In still another embodiment, this connection base has can cracking, to discharge the group of modified group from saccharide residue.Many cleavable moiety are well known in the art.Such as, see people such as Jung, Biochem.Biophys.Acta761:152-162 (1983); The people such as Joshi, J.Biol.Chem.265:14518-14525 (1990); The people such as Zarling, J.Immunol.124:913-920 (1980); The people such as Bouizar, Eur.J.Biochem.155:141-147 (1986); The people such as Park, J.Biol.Chem.261:205-210 (1986); The people such as Browning, J.Immunol.143:1859-1867 (1989).And, various cleavable, difunctionality (with Heterobifunctional) be connected base can from supplier such as Pierce there commercial.
Exemplary cleavable structure division can make to use up, heat or reagents ratio as mercaptan, azanol, alkali, periodate etc. carry out cracking.And, as the response to endocytosis, some preferred group cracking in vivo (such as, cis-rhizome of Chinese monkshood base; See people such as Shen, Biochem.Biophys.Res.Commun.102:1048 (1991)).The group of preferred cleavable comprises the structure division of cleavable, and it is selected from disulphide, ester, imide, carbonic ether, nitrobenzyl, the member in benzoyl and bitter almond oil camphor group.
Method
Present invention also offers the method for the basic single dispersing colony of preparing PEG molecule.The method comprises the PEG molecule allowing and have clear and definite molecular weight, the difunctionality activated PEG also with clear and definite molecular weight of such as PEG200 and at least 2 equivalents, such as PEG200 contact, thus forms the single dispersing sample of PEG, such as PEG600:
G is leavings group, such as sulphonate and tresylate ester.Then the single dispersing sample of PEG600 can contact with difunctionality activated PEG 200, forms single dispersing PEG100.Or single dispersing PEG600 can be converted into corresponding bifunctional derivative and react with the single dispersing dihydroxyl PEG600 of at least 2 equivalents, form single dispersing PEG1800.Till method of the present invention repeats to the single dispersing PEG obtaining desired size.This synthesis can be designed, make the molecular weight differences between starting raw material and product allow to be separated by size exclusion chromatography, the material of any unreacted or partial reaction.
Activated PEG derivative
Present invention also offers the method preparing PEG derivative.The method is summarised in scheme I:
A.R-Y/ (acid or alkali); B. activate, such as tosylation, halogen-go hydroxylation, such as HX or SOX 2, and and PEG mreaction; C. activate (R '), such as, use chloroformic acid p-nitrophenyl ester.
Wherein, subscript m and n represent 1-100 independently, the integer of 000.R is selected from replacement or non-substituted alkyl, replaces or does not replace assorted alkyl, replaces or unsubstituting aromatic yl, alkylamine, protection alkylamine, or the member in activating group, such as trifluoromethanesulfonic acid root, tosylate etc.
R ' is selected from replacement or non-substituted alkyl, replaces or does not replace assorted alkyl, replaces or unsubstituting aromatic yl, replaces or unsubstituting heterocycle alkyl and replacement or non-substituted heteroaryl.When R does not comprise the CH connected for activating it 2during the leavings group of-O structure division, R ' is generally leavings group, or comprises leavings group.
In an exemplary embodiment, R is low alkyl group, such as methyl.In another exemplary embodiment, R ' is substituted alkyl, such as chloroformic acid p-nitrophenyl ester.
In step a, original glycol contacts with activating group (R-Y), and the hydroxyl moiety of this activating group and glycol reacts.Y is leavings group normally, and R is placed on one of hydroxyl moiety of PEG molecule.In stepb, the group that the free hydroxyl group of gained adducts is converted into such as halogen such as chlorine or sulphonate and so on by it activates.Activation PEG material with as initial PEG (" PEG m") the identical or different polymerization degree another PEG structure division contact.Another material is connected to, RO-PEG in order to make it (n+m)optionally activate in free hydroxyl group structural portion office.
Generally, R group is connected to PEG structure division via the material comprising reactive functional groups.And two PEG fragments connect together by using reactive functional groups, and they are converted into new organo-functional group or unreacted material by method of attachment.Reactive functional groups is positioned at any position on PEG structure division, but preferably at one of end.
The sugar of branched polymer modification and puting together of peptide
Modified sugars uses suitable enzyme to put together in glycosylation or non-glycosylated peptide, puts together to reconcile this.Preferably, select modification to the concentration of body sugar, enzyme and acceptor peptide, glycosylation is proceeded to till acceptor is consumed.Although illustrate with regard to sialyltransferase, following consideration is generally applicable to the reaction of other glycosyltransferase.
The many methods using the required oligosaccharide structure of glycosyltransferase synthesis are known, and are generally applicable to the present invention.The people such as illustrative methods is such as described in WO96/32491, Ito, Pure Appl.Chem.65:753 (1993), and U.S.Pat.Nos.5,352,670,5,374,541, and 5,545,553.
The present invention uses the binding substances of single glycosyltransferase or glycosyltransferase to implement.Such as, the binding substances of sialyltransferase and galactosyltransferase can be used.In these embodiments of enzyme using more than one, enzyme and substrate preferably merge in initial reaction mixture, or once the first enzymatic reaction terminates or almost terminates, the enzyme and reagent that are used for the second enzymatic reaction are joined in reaction medium.By carrying out two kinds of enzymatic reactions according to the order of sequence in single container, the operation that total recovery compares wherein separation of intermediates material is improved.And, decrease removing and the process of added solvent and by product.
In preferred embodiments, first and second kinds of each glycosyltransferases naturally of enzyme.In another preferred embodiment, a kind of enzyme is interior Glycosylase.In a further preferred embodiment, use two or more enzymes to assemble modified proteins of the present invention.Before and after modified sugars is added to peptide, enzyme is used to change the sugared structure of any position on peptide.
The glycosyl structure part that the O-of conjugate of the present invention connects generally assigns to produce by the GalNAc structural portion being connected to peptide.Any member of the family of GalNAc transferring enzyme can be used for GalNAc structure division being incorporated into peptide (Hassan H, Bennett EP, Mandel U, Hollingsworth MA, and Clausen H (2000)).Mucus type O-glycosylation: the control of O-glycan occupation rate is instructed (Eds.Ernst, Hart, and Sinay) by the substrate specificity of polypeptide GalNAc-transferring enzyme.Wiley-VCH chapter Carbohydrates inChemistry and Biology-a Comprehension Handbook″，273-292)。GalNAc structure division itself can be that complete glycosyl connects base.In addition, glycosyl residue can use the glycosyl substrate of one or more enzymes and one or more suitable enzymes to increase and build, and modified sugars is added in the glycosyl structure part that increases and build.
In another embodiment, the method utilizes one or more outer or interior Glycosylases.Glycosylase is generally mutant, and it is formed glycosyl bond by through engineering approaches, but not cracking they.Mutant glycanase generally comprises the amino-acid residue replacing avtive spot acidic amino acid residue.Such as, when interior glycanase is interior-H, replacement active-site residues is generally the Asp at 130, at the Glu of 132, or their combination.Amino acid generally uses Serine, L-Ala, and l-asparagine or glutamine replace.
Mutant enzyme carrys out this reaction of catalysis by the synthesis step similar with the reversed reaction of interior glycanase hydrolysis step usually.In these embodiments, glycosyl donor molecule (such as, required oligosaccharides or monose structure) is containing leavings group, and this reaction is undertaken by being added to by donor molecule on the GlcNAc residue on albumen.Such as, leavings group can be halogen, such as fluorine.In other embodiments, leavings group is Asn, or Asn-peptide structure division.In other embodiments, the GlcNAc residue on glycosyl donor molecule is modified.Such as, GlcNAc residue can comprise 1,2- azoles quinoline structure division.
In preferred embodiments, the various enzymes for the production of conjugate of the present invention exist with catalytic amount.The catalytic amount of certain enzyme changes according to the concentration of the substrate of enzyme and reaction conditions such as temperature, time and pH value.The mode measuring the catalytic amount of set enzyme under preselected concentration of substrate and reaction conditions is as well known to those skilled in the art.
The temperature of carrying out above method can be the temperature of the temperature just more than freezing to the most responsive enzyme denaturation.Preferred temperature range is about 0 to about 55 DEG C, and is more preferably about 20 to about 30 DEG C.In another exemplary embodiment, one or more components of the inventive method at high temperature use Zimadzhunt L 340 process.
Reaction mixture keeps acceptor to be enough to be glycosylated, thus forms the time of required conjugate.Some of conjugate usually can detect after several hours, and recyclable amount reclaims usually in 24 hours or shorter time.Those skilled in the art will recognize that speed of reaction depends on the value (such as enzyme concn, to bulk concentration, acceptor density, temperature, solvent volume) of variable factor, these values can be optimized for selective system.
Present invention also offers the industrial-scale production method of modification peptide.Technical scale used herein generally creates at least approximately 250mg, finished product, the purification conjugate of preferred at least approximately 500mg and more preferably at least about 1g, preferably after a reaction time terminates, that is, this conjugate is not the binding substances of the reaction product of synthesis cycle from identical, continuous repetition.
In the following discussion, the present invention is by illustrating modification sialic acid structure part and puting together of glycosylated peptide.Exemplary modification sialic acid (m-) PEG marks.Below discussing the use concentrating on PEG modification sialic acid and glycosylated peptide is to clearly demonstrate, and is not the conjugate that hint the present invention is limited to these two kinds of counterparts.Technician understands, and this discussion is generally applicable to the addition of the modified sugars based moiety except sialic acid.And this discussion is equally applicable to the modification of glycosyl units with the reagent (comprising other water-soluble polymers, treatment structure division and biomolecules) except PEG.
Enzymatic means may be used for the carbohydrate selectivity of branched polymer modification to be incorporated on peptide or glycopeptide.The method adopts the modified sugars containing branched water-soluble polymers, and is combined with suitable glycosyltransferase or sugared synthetic enzyme.By selecting to obtain the glycosyltransferase of required carbohydrate key and adopting modified sugars as to body substrate, this branched water-soluble polymers can be introduced directly on peptide backbone, on the existing saccharide residue of glycopeptide or on the saccharide residue having added on peptide.
The acceptor of sialyltransferase is as naturally occurring structure, or as restructuring, the structure that enzyme process or chemical method are arranged be present in will by method modification peptide of the present invention, the acceptor be applicable to such as comprises galactosyl acceptor, such as GalNAc, Gal β 1, 4GlcNAc, Gal β 1, 4GalNAc, Gal β 1, 3GalNAc, LNT, Gal β 1, 3GlcNAc, Gal β 1, 3Ara, Gal β 1, 6GlcNAc, Gal β 1, 4Glc (lactose), and other acceptor well known by persons skilled in the art is (for example, see people such as Paulson, J.Biol.Chem.253:5617-5624 (1978)).
In one embodiment, the acceptor of sialyltransferase is present on the glycopeptide of modification when synthesizing in the body of glycopeptide.It is sialylated that this type of glycopeptide can use required method, and do not answer preliminary election to change the glycosylation patterns of glycopeptide.Or method of the present invention can be used for the sialylated peptide not comprising applicable acceptor; First people can pass through this peptide of method known to those skilled in the art modification, to introduce acceptor.
In an exemplary embodiment, galactosyl acceptor is by being connected to the suitable acceptor be connected with peptide by galactose residue, such as Ga lNAc assembles.The method comprise with containing appropriate galactosyltransferase (such as Gal β 1,3 or Gal β Isosorbide-5-Nitrae) and the galactosyl that is applicable to the reaction mixture of body (such as UDP-semi-lactosi) hatch want the peptide of modification.This reaction is proceeded to substantially complete, or, when with the addition of the galactose residue of predetermined amount, termination reaction.Assembling selects other method of saccharide acceptor to be that those skilled in the art is apparent.
In still another embodiment, the oligosaccharides that first overall or part " pruning " glycopeptide connects, to expose the acceptor of sialyltransferase, or can add one or more applicable residue to obtain the structure division of applicable acceptor.Enzyme such as galactosyltransferase and endoglycosidase (for example, see US patent No.5,716,812) can be used for connecting and pruning reaction.
In an exemplary embodiment, carbohydrate residue is before interpolation modified sugars " pruning ".Such as, GalNAc-Gal residue is trimmed to GalNAc again.The modified sugars of carrying water-soluble polymers puts together the one or more saccharide residues in being exposed by " pruning ".In an example, " pruning " glycopeptide, such as, via the glycosyl structure part of puting together in water-soluble polymers, Sia, Gal or GalNAc structure division, adds to water-soluble polymers on gained O-side chain amino acid or glycopeptide glycan.Modified sugars based moiety is connected on the acceptor site on the glycopeptide of " pruning ".Such as, or unmodified glycosyl structure part, the glycan end that Gal can connect at O-connects.
In another exemplary embodiment, via the modified sugars with galactose residue, branched water-soluble polymers is added on GalNAc residue.Or, unmodified Gal can be added to end GalNAc residue.
In another example, modification sialic acid is used to be added on Gal residue by branched water-soluble polymers.
Above-mentioned exemplary provides the explanation of the ability of method as herein described.Use method of the present invention, can " cut down " and the carbohydrate residue of " increase " any desirable configuration substantially.Modified sugars can add to the end of carbohydrate moiety as above, or it can between peptide core and carbohydrate end.
In an exemplary embodiment, the sialic acid of polymer modification is used to be added on terminal Gal residue by branched water-soluble polymers.Suitable sialyltransferase is used to carry out addition modification sialic acid.The method is summed up in scheme 3.
Scheme 3:
In another method summed up in scheme 4, the reactive functional groups closed is present in sialic acid.This capping group is not preferably exposed for the impact of condition modification sialic acid being connected to peptide.After modification sialic acid is covalently attached to peptide, removing is covered and is closed thing, uses the reagent of such as PEG, PPG, treatment structure division, biomolecules and so on or other reagent to put together peptide.This reagent is puted together in peptide in a specific way by the reaction of the non-capping group on itself and modified sugars residue.
Scheme 4
Depend on the end sugar (table 3) of the oligosaccharide side chains of glycopeptide, any modified sugars can use together with its suitable glycosyltransferase.As mentioned above, the end sugar introducing the glycopeptide needed for branched water-soluble polymers structure naturally can be introduced in expression process, or it can pass through to use suitable Glycosylase, glycosyltransferase, or the mixture of Glycosylase and glycosyltransferase generates after expression.
Table 3
In alternative embodiment, the glycosyltransferase on the glycosylation site using known O-glycosyl residue being transferred to peptide backbone to connect, by modified sugars direct addition on peptide backbone.This exemplary is set forth in scheme 5.Can be used for implementing exemplary glycosyltransferase of the present invention including, but not limited to GalNAc transferring enzyme (GalNAc T1-20), GlcNAc transferring enzyme, fucosyltransferase, glucanotransferase, xylosyltransferase, mannose transferase etc.Use the method can by the peptide of modified sugars direct addition to any carbohydrate of shortage, or on existing glycopeptide.In both cases, the addition of modified sugars occurs in as on the specific position on the peptide backbone of the substrate specificity defined by glycosyltransferase, instead of resembles the random fashion occurred in the peptide backbone process making chemically modified protein.By by suitable aminoacid sequence through engineering approaches in polypeptide chain, reagents series can be incorporated in the albumen or glycopeptide lacking glycosyltransferase substrate peptide sequences.
Scheme 5
In above-mentioned each exemplary, modified sugars is being puted together after peptide, one or more additional chemical or enzyme modification step can adopted.In an exemplary embodiment, enzyme (such as fucosyltransferase) is used glycosyl units (such as Fucose) to be connected to the terminal-modified sugar be connected with peptide.In another example, the site that next " end-blocking " (such as sialylated) modified sugars of enzymatic reaction fails to put together is used.Or, adopt chemical reaction to change the structure of the modified sugars of puting together.Such as, the modified sugars of puting together and the reagent react stablizing or go the key stablizing the peptide composition that it is connected with modified sugars.In another example, put together after peptide at it, the component of modified sugars is gone protection.Technician is clear, modified sugars is being puted together in the stage after peptide, and the serial enzymes with method used in the present invention is urged to become reconciled chemical process.The further details of modified sugars-peptide conjugate is within the scope of the invention.
In another exemplary embodiment, puted together by glycopeptide in directed agents, such as siderophilin (for carrying peptide to pass hemato encephalic barrier, and arriving endosome), carnitine is (for being transported to muscle cell by peptide; For example, see people such as LeBorgne, Biochem.Pharmacol.59:1357-63 (2000), and phosphonic acid ester, such as bisphosphonate is (for being directed to bone and other calcic tissue by peptide; For example, see Modern Drug Discovery, in August, 2002, the 10th page).Other reagent that can be used for leading is that those skilled in the art institute is apparent.Such as, glucose, glutamine and I GF also can be used for being directed to muscle.
Targeting moiety and therapeutic peptide are puted together by any method as herein described or other method known in the art.Technician is clear, and peptide in addition to those described above also can derivatize as described herein.At unexamined, the US temporary patent application No.60/328 that owns together that October 10 calendar year 2001 submits to, in the annex appended by 523, set forth exemplary peptides.
In an exemplary embodiment, directed agents and therapeutic peptide are via being connected based moiety coupling.In this embodiment, according to method of the present invention, at least one of therapeutic peptide or directed agents connects base via complete glycosyl and is coupled to connection based moiety.In an exemplary embodiment, connect based moiety and comprise poly-(ether) such as PEG.In another exemplary, after connection based moiety is included in the destination organization or position conjugate being transported to human body, degradation in vivo, from least one key of directed agents release therapeutic peptide.
In another exemplary, therapeutic peptide, by changing the sugared shape for the treatment of structure division, need not be puted together and change in targeting moiety by the distribution in vivo of therapeutic peptide.Such as, therapeutic peptide can by avoid with the terminal galactose structure division of sialic acid (or derivatives thereof) end-blocking glycosyl absorb by reticuloendothelial system.
I. enzyme
1, glycosyltransferase
Glycosyltransferase is catalytic activation sugar (to the body NDP-sugar) addition on albumen, glycopeptide, lipid or glycolipid or on the non-reducing end of growth oligosaccharides in a step-wise fashion.The oligosaccharides that the glycopeptide that N-connects is connected with lipid via transferring enzyme is to body Dol-PP-NAG 2glc 3man 9synthesize with global transfer and the mode of pruning core subsequently.In this case, the character of " core " sugar is different from follow-up connection a bit.Known in the art have a large amount of glycosyltransferases.
Can be any one for glycosyltransferase of the present invention, as long as it can use modified sugars as sugar to body.The example of this fermentoid comprises Leloir approach glycosyltransferase, such as galactosyltransferase, NAG based transferase, N-acetylamino galactosamine based transferase, fucosyltransferase, sialyltransferase, mannose transferase, xylosyltransferase, glucuronyl transferase etc.
For the enzymatic sugar synthesis relating to glycosyltransferase reaction, glycosyltransferase can be cloned, or is separated from any source.It is known for being permitted polyclonal glycosyltransferase, and their polynucleotide sequence is also known.For example, see " The WWW Guide To ClonedGlycosyltransferases ", ( http:// www.vei.co.uk/TGN/gt-guide.htm).Glycosyltransferase aminoacid sequence also can find with the nucleotide sequence of the encoding glycosyltransferases can reasoning out aminoacid sequence in the obtainable database of the various public, comprises GenBank, Swiss-Prot, EMBL etc.
The glycosyltransferase that can use in the method for the invention is including, but not limited to galactosyltransferase, fucosyltransferase, glucanotransferase, N-acetylamino galactosamine based transferase, NAG based transferase, glucuronyl transferase, sialyltransferase, mannose transferase, glucuronyl transferase, galacturonic acid transferring enzyme and oligosaccharyl transferase.The glycosyltransferase be applicable to comprises those that obtained by eukaryotic cell and prokaryotic organism.
The glycosyltransferase of DNA encoding can pass through chemosynthesis, screens the reverse transcription product of mRNA from suitable cell or cloned culture, by the gene pool of screening from suitable cell, or by obtaining in conjunction with these operations.The screening of mRNA or genomic dna can be carried out with the oligonucleotide probe produced by glycosyltransferase gene sequence.Probe can according to operation detectable group such as fluorophor that is known and that use in standard hybridization test, and radioactive atom or chemiluminescent groups mark.In the scheme substituted, glycosyltransferase gene sequence can obtain by using polymerase chain reaction (PCR) operation, and wherein PCR Oligonucleolide primers is produced by glycosyltransferase gene sequence.See US patent No.4,683,195 (people such as Mullis) and US patent No.4,683,202 (Mullis).
Glycosyltransferase can synthesize in the host cell of the vector with the DNA containing encoding glycosyltransferases.Carrier is used for the DNA of amplification coding glycosyltransferase and/or expresses the DNA of encoding glycosyltransferases.Expression vector is reproducible DNA structure, and wherein the DNA sequence dna of encoding glycosyltransferases is operably connected to the applicable control sequence (control sequence) can expressing this glycosyltransferase in applicable host.Demand for this control sequence changes according to selected host and selected method for transformation.Generally, control sequence comprises transcripting promoter, optional for controlling the manipulation group sequence of transcribing, the sequence of the mRNA ribosome binding site that coding is applicable to, and controls the sequence of the termination of transcribing and translating.Amplification vector does not need to express control region.Required whole are the abilities copied in host, usually given by the Select gene of the starting point copied and the identification that is conducive to transformant.
In an exemplary embodiment, the present invention adopts protokaryon enzyme.This glycosyltransferase is included in the enzyme related in the synthesis of fat oligosaccharides (LOS), they produce people such as (, Critical Reviews in Microbiology23 (3): 139-180 (1996)) Preston by many gram negative bacteriums.This fermentoid is including, but not limited to the albumen of the rfa operon of species such as E.coli and Salmonella typhimurium, they comprise β-1,6-galactosyltransferase and β-1,3-galactosyltransferase is (such as, see EMBL Accession Nos.M80599 and M86935 (E.coli); EMBL Accession No.S56361 (S.typhimurium)), glycosyltransferase (Swiss-Prot Accession No.P25740 (E.coli), β 1,2-glucanotransferase (rfaJ) (Swiss-Prot Accession No.P27129 (E.coli) and Swiss-Prot Accession No.P 19817 (S.typhimurium)), with β 1,2-N-acetylamino glucosyl transferring enzyme (rfaK) (EMBL Accession No.U00039 (E.coli)).Other glucanotransferase that aminoacid sequence is known comprise by operon such as rfaB (they are at microorganism such as klebsiella pneumoniae, intestinal bacteria, bacillus typhi murium, intestines Salmonellas (Salmonella enteric), Yersinia enterocolitica, qualification in Mycobacterium leprae (Mycobacterium leprosum)), and those of the rhl operon coding of Pseudomonas aeruginosa.
Also being applicable to of the present invention is in production containing Lacto-N-neo-tetraose (lacto-N-neotetraose), D-galactosyl-β-Isosorbide-5-Nitrae-N-ethanoyl-GLUCOSAMINE base-β-1,3-D-galactosyl-β-Isosorbide-5-Nitrae-D-Glucose, and P kblood group three glycosylation sequence, D-galactosyl-α-1,4-D-galactosyl-β-1, the glycosyltransferase related in the structure of 4-D-glucose, their qualifications in the LOS of mucosal pathogen Diplococcus gonorrhoeae (Neisseria gonnorhoeae) and Neisseria meningitidis (N.meningitidis) people such as (, J.Med.Microbiol.41:236-243 (1994)) Scholten.The Neisseria meningitidis of the encoding glycosyltransferases related in the biosynthesizing of these structures and the gene of Diplococcus gonorrhoeae are by Neisseria meningitidis immunologic pattern (immunotypes) the L 3 and L1 (people such as Jennings, Mol.Microbiol.18:729-740 (1995)) and Diplococcus gonorrhoeae mutant F62 (Gotshlich, J.Exp.Med.180:2181-2190 (1994)) identification.In Neisseria meningitidis, by three gene lgtA, the loci encode of lgtB and lgE composition adds glycosyltransferase needed for last three sugar people such as (, J.Biol.Chem.271 (45): 19166-73 (1996)) Wakarchuk in Lacto-N-neo-tetraose (lacto-N-neotetraose).Recently, the enzymatic activity of lgtB and IgtA gene product is proved, provide first direct evidence (people such as Wakarchuk, J.Biol.Chem.271 (45): 28271-276 (1996)) of they proposed glycosyltransferase function.In Diplococcus gonorrhoeae, there are two other genes, β-D-GalNAc added to the lgtD on 3 of the terminal galactose of Lacto-N-neo-tetraose (lacto-N-neotetraose) structure and end α-D-Gal added to the lactose structural unit blocking LOS, therefore creating P kthe lgtC of blood group antigen structure (Gotshlich (1994) above).In Neisseria meningitidis, independently immunologic pattern L1 also have expressed P kblood group antigen, and shown to carry lgtC gene (people such as Jennings, (1995), above).Neisseria glycosyltransferase and genes involved are also described in USPN 5, and 545, in 553 (Gotschlich).From α 1, the 2-fucosyltransferase of helicobacter pylori and α 1,3-fucosyltransferase gene also identified (people such as Martin, J.Biol.Chem.272:21349-21356 (1997)).Also used in the present invention is the glycosyltransferase (for example, see http://afmb.cnrs-mrs.fr/ ~ pedro/CAZY/gtf_42.html) of campylobacter jejuni.
A) fucosyltransferase
In certain embodiments, the glycosyltransferase for method of the present invention is fucosyltransferase.Fucosyltransferase is known to those skilled in the art.Exemplary fucosyltransferase comprises the enzyme of hydroxy position L-fucose being transferred to acceptor saccharide from GDP-Fucose.The fucosyltransferase that acceptor transferred to by non-nucleotide sugar also be can be used for the present invention.
In certain embodiments, acceptor saccharide be such as in oligosaccharides glucosides Gal β (1 → 3,4) GlcNAc beta-yl group in GlcNAc.The fucosyltransferase being applicable to this reaction comprises Gal β (1 → 3,4) GlcNAc β 1-α (1 → 3,4) fucosyltransferase (FTIIIE.C.No.2.4.1.65), they are first identified from human milk (see people such as Palcic, Carbohydrate Res.190:1-11 (1989); The people such as Prieels, J.Biol.Chem.256:10456-10463 (1981); And the people such as Nunez, Can.J.Chem.59:2086-2095 (1981)), and Gal β (1 → 4) GlcNAc β-α fucosyltransferase (FTIV, FTV, FTVI), they are found in human serum.((1 → 3) GlcNAc β fucosyltransferase is also identified for FTVII (E.C.No.2.4.1.65), saliva acidic group α (2 → 3) Gal β.Gal β (1 → 3,4) GlcNAc β-α (1 → 3,4) recombinant forms of fucosyltransferase is also identified (see people such as Dumas, the people such as Bioorg.Med.Letters 1:425-428 (1991) and Kukowska-Latallo, Genes andDevelopment 4:1288-1303 (1990)).Other exemplary fucosyltransferase such as comprises α 1,2-fucosyltransferase (E.C.No.2.4.1.69).Enzymatic fucosylation can pass through people such as Mollicone, Eur.J Biochem.191:169-176 (1990) or U.S. patent No.5, and 374, the method described in 655 is carried out.Cell for the production of fucosyltransferase also comprises the enzymatic system for the synthesis of GDP-Fucose.
B) galactosyltransferase
In another group embodiment, the galactosyltransferase of glycosyltransferase.Exemplary galactosyltransferase comprises α (1,3) galactosyltransferase (E.C.No.2.4.1.151, for example, see people such as Dabkowski, the people such as Transplant Proc.25:2921 (1993) and Yamamoto, Nature 345:229-233 (1990), ox (GenBank j04989, the people such as Joziasse, J.Biol.Chem.264:14290-14297 (1989)), murine (GenBank m26925; The people such as Larsen, Proc.Nat ' l.Acad.Sci.USA86:8227-8231 (1989)), pig (GenBank L36152; The people such as Strahan, Immunogenetics 41:101-105 (1995)).Another α 1,3 galactosyltransferase be applicable to is that (EC 2.4.1.37, the people such as Yamamoto, J.Biol.Chem.265:1146-1151 (1990) (people)) that relate in the synthesis of blood group B antigen.Another exemplary galactosyltransferase is core Gal-T1.
That be also applicable to method of the present invention is β (1, 4) galactosyltransferase, such as comprise EC2.4.1.90 (LacNac synthetic enzyme) and EC2.4.1.22 (lactose synthetase) (ox (people such as D ' Agostaro, Eur.J.Biochem.183:211-217 (1989)), people (the people such as Masri, Biochem.Biophys.Res.Commun.157:657-663 (1988)), mouse (the people such as Nakazawa, J.Biochem.104:165-168 (1988)), and E.C.2.4.1.38 and ceramide galactosyl transferring enzyme (EC 2.4.1.45, the people such as Stahl, J.Neurosci.Res.38:234-242 (1994)).Other galactosyltransferase be applicable to such as comprises α 1,2 galactosyltransferases (such as from people such as Schizosaccharomycespombe, Chapell, Mol.Biol.Cell 5:519-528 (1994)).
C) sialyltransferase
Sialyltransferase is the another kind of glycosyltransferase that can be used for reconstitution cell and reaction mixture of the present invention.The cell of Restruction sialyltransferase also produces cmp sialic acid, and it is that the sialic acid of sialyltransferase is to body.The example being applicable to sialyltransferase of the present invention comprises ST3Gal III (such as mouse or people ST3Gal III), ST3Gal IV, ST3GalI, ST6Gal I, ST3Gal V, ST6Gal II, ST6Gal NAc I, ST6GalNAc II, and ST6GalNAc III (in sialyltransferase nomenclature used herein as people such as Tsuji, described in Glycobiology 6:v-xiv (1996)).Be called that sialic acid is transferred on the non-reducing end Gal of Gal β 1 → 3 Glc disaccharides or glucosides by exemplary α (2, the 3) sialyltransferase of α (2,3) sialyltransferase (EC2.4.99.6).See people such as Van denEijnden, J.Biol.Chem.256:3159 (1981), the people such as Weinstein, the people such as J.Biol.Chem.257:13845 (1982) and Wen, J.Biol.Chem.267:21011 (1992).Sialic acid is transferred on the non-reducing end Gal of disaccharides or glucosides by another exemplary α 2,3-sialyltransferase (EC2.4.99.4).See people such as Rearick, the people such as J.Biol.Chem.254:4444 (1979) and Gillespie, J.BioL Chem.267:21004 (1992).Other exemplary enzyme comprises Gal-β-Isosorbide-5-Nitrae-GlcNAc α-2,6 sialyltransferase (see people such as Kurosawa, Eur.J.Biochem.219:375-381 (1994)).
Preferably, for the glycosylation of the carbohydrate of glycopeptide, sialic acid can be transferred to sequence Gal β 1 by sialyltransferase, on 4GlcNAc-, be positioned at the sequence modal second from the bottom (see table 4) below the terminal sialic acid on complete sialylated carbohydrate structure.
Table 4: use Gal β Isosorbide-5-Nitrae GlcNAc sequence as the sialyltransferase of receptor substrate
1) people such as Goochee, Bio/Technology 9:1347-1355 (1991)
2) people such as Yamamoto, J.Biochem.120:104-110 (1996)
3) people such as Gilbert, J.Biol Chem.271:28271-28276 (1996)
The example that can be used for the sialyltransferase of required method is ST3Gal III, is also called α (2,3) sialyltransferase (EC2.4.99.6).This enzyme catalysis sialic acid transfer to Gal β 1,3GlcNAc or Gal β Isosorbide-5-Nitrae GlcNAc Gal (for example, see people such as Wen, J.Biol.Chem.267:21011 (1992); The people such as Van den Eijnden, J.Biol.Chem.256:3159 (1991)), and the oligosaccharides causing the l-asparagine in glycopeptide to connect is sialylated.This sialic acid is connected to Gal, defines the α key between two sugar.Keyed jointing (connection) between sugar is between 2 and 3 of Ga l of NeuAc.This specific enzyme can be separated from mouse liver (people such as Weinstein, J.Biol.Chem.257:13845 (1982)); People cDNA (people (1993) such as Sasaki, J.Biol.Chem.268:22782-22787; Kitagawa & Paulson (1994), and the genome (people such as Kitagawa J.Biol.Chem.269:1394-1401), (1996) J.Biol.Chem.271:931-938) DNA sequence dna is known, is conducive to producing this enzyme by recombinant expressed.In a preferred embodiment, required sialylation methods uses mouse ST3Gal III.
Comprise for other exemplary sialyltransferase of the present invention those that be separated from campylobacter jejuni, comprise α (2,3).For example, see WO99/49051.
Except enumerate in table 5 those except sialyltransferase also can be used for the economy of sialylated industrial important glycopeptide and effective large-scale methods.As the simple experiment of the purposes of these other enzymes of discovery, each enzyme (1-100mU/mg albumen) of different amount reacts with de-sialic acid-α 1AGP (1-10mg/ml), to compare the ability of the sialylated glycopeptide of studied sialyltransferase and ox ST6Gal I, ST3GalIII or these two kinds of glycosyltransferases.Or the oligosaccharides that other glycopeptide discharged from peptide backbone with enzyme process or N-connect can replace de-sialic acid-α 1AGP for this evaluation.The sialyltransferase of ability with the oligosaccharides connected than the N-of ST6Gal I more effectively sialylated glycopeptide can be used for the sialylated practical large-scale methods of peptide (as in the disclosure to illustrated by ST3Gal III).
D) GalNAc transferring enzyme
GalNAc based transferase can be used for implementing the present invention, the amino acid of the glycosylation site that the O-being particularly useful for GalNAc structure division to be incorporated into peptide connects.The GalNAc based transferase be applicable to is including, but not limited to α (1; 3) N-acetyl galactose based transferase; β (1; 4) the GalNAc based transferase (people such as Nagata; the people such as J.Biol.Chem.267:12082-12089 (1992) and Smith; J.Biol Chem.269:15162 (1994)) and polypeptide GalNAc based transferase (people such as Homa, J.Biol.Chem.268:12609 (1993)).
By genetically engineered by the genes produce protein ratio of cloning as enzyme GalNAcT 1-XXmethod be known.For example, see US patent No.4,761,371.Method comprises collects enough samples, is then measured the aminoacid sequence of enzyme by N-end sequencing.Then use this information to be separated the cDNA clone of encoding full leng (film combination) transferring enzyme, when it expresses in insect cell line Sf9, synthesize complete organized enzyme.Then the amino acid whose semi-quantitative analysis around the known glycosylation site being used in 16 kinds of different albumen, carries out the external glycosylation research of synthetic peptide subsequently to measure the receptor-specific of enzyme.This research work is verified, some amino-acid residue excessively shows in glycosylated peptide segment, and the residue in specific position around glycosylation Serine and threonine residues can have more remarkably influenced than other amino acid structure part to acceptor efficiency.
2, sulfotransferase
Present invention also offers production and comprise sulfating numerator, such as sulfation polypeptide such as heparin, Suleparoid, the method for the peptide of carrageenin (carragnen) and related compound.Be applicable to sulfotransferase comprise such as chrondroitin-6-sulfotransferase (by people such as Fukuta, the chicken cDNA described in J.Biol.Chem.270:18575-18580 (1995); GenBank Accession No.D49915), glycosaminoglycan NAG N-deacetylase/N-sulfotransferase 1 (people such as Dixon, Genomics 26:239-241 (1995); UL 18918), and glycosaminoglycan NAG N-deacetylase/N-sulfotransferase 2 is (people such as Orellana, the people such as J.Biol.Chem.269:2270-2276 (1994) and Eriksson, the murine cDNA described in J.Biol.Chem.269:10438-10443 (1994); People cDNA described in GenBankAccession No.U2304).
3, the glycosyltransferase of Cell binding
In another embodiment, the enzyme adopted in the method for the invention is the glycosyltransferase of Cell binding.Although many soluble sugar based transferases are known (for example, see US patent No.5,032,519), when with cell communication, glycosyltransferase exists with film combining form usually.Many membrane bound enzymes of studying are considered to intrinsic protein so far; That is, they do not disengage from film by supersound process, need the washing composition for solubilising.Surface glycosyltransferase is identified on the surface of vertebra and invertebral zooblast, also has realized that these surperficial transferring enzymes maintain catalytic activity in physiological conditions.But the function of more generally acknowledged cell surface glycosyltransferase is iuntercellular identification (Roth, MOLECULAR APPROACHES toSUPRACELLULAR PHENOMENA, 1990).
Develop method to change by the glycosyltransferase of cell expressing.Such as, the people such as Larsen, Proc.Natl.Acad.Sci.USA 86:8227-8231 (1989), reports and measures cell surface oligosaccharide structure and their genetic method associating the cDNA sequence of the separating clone of the expression of glycosyltransferase.Will by known expression UDP-galactase: the generation cDNA library of the mRNA that the murine cells system of β-D-galactosyl-Isosorbide-5-Nitrae-N-ethanoyl-D-glucosaminide α-1,3-galactosyltransferase is separated is transfected into COS-1 cell.Then cultivate the cell of transfection, and analyze α-1,3-galactosyltransferasactivity activity.
The people such as Francisco, Proc.Natl.Acad.Sci.USA 89:2713-2717 (1992) discloses method β-lactamase being anchored in colibacillary outside surface.Produce by the signal sequence of (i) outer membrane protein, (ii) the cross-film section of outer membrane protein, (iii) three syzygys of full ripe β-lactamase sequence composition, obtain the β-lactamase molecule that active surface combines.But Francisco method is only limitted to prokaryotic cell prokaryocyte system, according to the understanding of author, suitable playing a role needs complete three to merge.
4, fusion rotein
In other exemplary, method of the present invention adopts the fusion rotein with more than one the enzymic activity relevant with the synthesis of desired sugars peptide conjugate.Fusion polypeptide can such as be made up of the catalytic activity region of glycosyltransferase in the catalytic activity region being connected to auxiliary enzymes.Auxiliary enzymes catalysis region such as can catalysis belong to glycosyltransferase to the step in the formation of the nucleotide sugar of body, or the reaction related in catalysis glycosyltransferase cycles.Such as, the polynucleotide of encoding glycosyltransferases can be connected to the polynucleotide being coded in the enzyme related in nucleotide sugar synthesis in frame.Gained fusion rotein then not only can the synthesis of catalysis nucleotide sugar, and catalysis sugar structure division is transferred on acceptor molecule.This fusion rotein can be connected to the two of an effable nucleotide sequence or multi cycle enzyme.In other embodiments, fusion rotein comprises the catalytic activity region of two or more glycosyltransferases.For example, see 5,641,668.Modification glycopeptide of the present invention can easily utilize various applicable fusion rotein to design and produce (such as, see PCT patent application PCT/CA98/01180, it is open on June 24th, 1999 as WO99/31224).
5, immobilized enzyme
Except the enzyme of Cell binding, present invention also offers the purposes of the enzyme be fixed in solid and/or soluble carrier.In an exemplary embodiment, provide method according to the present invention to connect base via intact glycosyl and put together glycosyltransferase in PEG.PEG-connects base-enzyme conjugate and is optionally connected to solid carrier.The enzyme that solid supports application in the method for the invention simplifies the aftertreatment of reaction mixture and the purification of reaction product, easily can also reclaim enzyme.Glycosyltransferase conjugate adopts in the method for the invention.Other combination of enzyme and carrier is that those skilled in the art institute is apparent.
The purification of peptide conjugate
The product produced in order to upper method can be purified and be used.But, usually preferably reclaim product.The standard for reclaiming glycosylation sugar, known technology such as thin layer or thick layer chromatography, column chromatography, ion exchange chromatography or membrane filtration can be adopted.Preferred use membrane filter method, more preferably adopts reverse osmosis membrane, or as hereafter with one or more Column chromatography techniques for reclaiming described in this paper citing document.Such as, wherein film has about 3000 to about 10, and the membrane filtration of the weight shutoff of 000 may be used for removing protein ratio as glycosyltransferase.Then nanofiltration or reverse osmosis can be used except desalting and/or purified product sugar (for example, see WO 98/15581).Nano-filtration membrane is by monovalent salt, but reservation is greater than about 100 to about 2, and a class reverse osmosis membrane of 000 daltonian a polyvalent salt and uncharged solute, this depends on used film.Therefore, in typical applications, be retained in film with sugar prepared by method of the present invention, but the salt polluted will pass through.
If modified proteins produces in molecule, as the first step, such as, by centrifugal or ultrafiltration removing granular debris, i.e. host cell or dissolving fragment, optionally, this albumen can concentrate with commercial protein concentration filter, subsequently by be selected from following among one or more step isolated polypeptide variant and other impurity: immunoaffinity chromatography, ion exchange column classification (such as using diethylamino ethyl (DEAE) or the matrix containing carboxymethyl or sulfapropyl), with Blue-Sepharose, CM Blue-Sepharose, MONO-Q, MONO-S, lens culinaris agglutinin (lentil lectin)-Sepharose, WGA-Sepharose, Con A-Sepharose, Ether Toyopearl, Butyl Toyopearl, Phenyl Toyopearl, SP-Sepharose, or protein A Sepharose chromatography, SDS-PAGE chromatography, silica gel chromatography, chromatofocusing, reversed-phase HPLC (such as there is the silica gel of Additional aliphatic base), such as use the gel-filtration of Sephadex molecular sieve or size exclusion chromatography, by the chromatography of the pillar of selective binding polypeptide, and ethanol or ammonium sulfate precipitation.
The modification glycopeptide produced in culture usually by initially extracting from cell, enzyme etc., and subsequently one or more concentrate, saltout, aqueous ionic exchanges or size exclusion chromatography step (such as SP Sepharose) is separated.In addition, modified proteins can be purified by affinity chromatography.HPLC can also be used for one or more purification step.
Proteinase inhibitor, such as methanesulfonyl fluoride (PMSF) in officely can how be gone up step and comprise, and with arrestin hydrolysis, and can comprise microbiotic, to prevent the growth of external contaminant.
In another embodiment, first the supernatant liquor from the system producing modification glycopeptide of the present invention uses commercial protein concentration filter, and such as Amicon or Millipore Pellicon ultra filtration unit concentrates.After enrichment step, this enriched material can be put on applicable purification matrix.Such as, the affinity matrix be applicable to can comprise the part of peptide, lectin or the antibody molecule being incorporated into applicable carrier.Or, can anionite-exchange resin be used, such as, there is matrix or substrate that side hangs DEAE group.Be applicable to matrix comprise acrylamide, agarose, dextran, Mierocrystalline cellulose, or in protein purification other type normally used.Or, can cation-exchange step be used.The cationite be applicable to comprises the various insoluble matrix containing sulfapropyl or carboxymethyl.Sulfapropyl is particularly preferred.
Finally, adopt hydrophobic RP-HPLC media, such as, there are one or more RP-HPLC steps that side hangs the silica gel of methyl or other aliphatic group and can be used for polypeptide variants composition of purifying further.Some or all of the above-mentioned purification step of various combination can also be used to the glycoprotein providing even modification.
The modification glycopeptide of the present invention obtained by large scale fermentation can by by people such as Urdal, and those the similar methods disclosed in J.Chromatog.296:171 (1984) are purified.This reference describes that by two of preparative HPLC pillar purification recombinant human il-2 RP-HPLC steps according to the order of sequence.In addition, the technology of such as affinity chromatography and so on can be used to purifying modified glycoprotein.
Except above-mentioned conjugate, the invention provides the method for these and other conjugate of preparation.And, the invention provides the method by conjugate for drug delivery of the present invention being prevented, treat or improved morbid state in the individuality or patient with disease risk.
Pharmaceutical composition
The treatment structure division puted together in branched water-soluble polymers of the present invention has various medicinal application.Such as modification erythropoietin (EPO) may be used for treating general anemia, aplastic anemia, the damage (such as to the damage of marrow) of chemotherapy induction, chronic renal failure, ephritis, and thalassemia.Modification EPO can be further used for treatment neurological disorder such as brain/Spinal injury, multiple sclerosis and alzheimer's disease.
Second example is interferon-alpha (IFN-α), it can be used for treatment AIDS and hepatitis B or the third liver, by various virus such as human papillomavirus (HBV), coronavirus, human immunodeficiency virus (HIV), herpes simplex virus (HSV), with the virus infection that varicella zoster virus (VZV) causes, cancer is hairy cell leukemia such as, AIDS dependency Kaposi sarcoma, malignant melanoma, follicularis non-hodgkin lymphoma, Philadelphia chromosome (Ph) is positive, chronic myelocytic leukemia (CML), kidney, myelomatosis, chronic myelogenous leukemia, tumor of head and neck, osteocarcinoma, and cervical dysplasia and central nervous system (CNS) disease such as multiple sclerosis.In addition, can be used for treating various Other diseases and state such as dry syndrome (autoimmune disease) according to the IFN-α of method modification of the present invention, behcet's disease (autoimmune inflammatory disease), fibromyalgia (flesh and skeleton pain/fatigue diseases), aphthous ulcer (canker sore), chronic fatigue syndrome, and pulmonary fibrosis.
Another example is beta-interferon, can be used for treatment CNS obstacle such as multiple sclerosis (relapsing/mitigation type or chronic progressive external), AIDS and hepatitis B or the third liver, by various virus such as human papillomavirus (HBV), human immunodeficiency virus (HIV), herpes simplex virus (HSV), with the virus infection that varicella zoster virus (VZV) causes, otic infections, flesh and infection of bone, and cancer, comprise mammary cancer, the cancer of the brain, colorectal carcinoma, nonsmall-cell lung cancer, incidence cancer, rodent cancer, cervical dysplasia, melanoma, skin carcinoma, and liver cancer.Other diseases and state such as transplant rejection (such as bone marrow transplantation) is also used for the treatment of, Huntington's chorea, colitis, encephalitis, pulmonary fibrosis, macular degeneration, liver cirrhosis, and keratoconjunctivitis according to the IFN-β of method modification of the present invention.
Granulocyte colony-stimulating factor (G-CSF) is another example.G-CSF according to method modification of the present invention can be used as auxiliary agent in the chemotherapy for the treatment of cancer, and such as, for preventing or alleviate the state of an illness relevant with some medical procedure or complication, the bone marrow injury of chemotherapy induction; Leukopenia (generality); The heat generation Neutrophilic granulocytopenia of chemotherapy induction; The Neutrophilic granulocytopenia relevant with bone marrow transplantation; And heavy chronic Neutrophilic granulocytopenia.Modification G-CSF also can be used for transplanting; Peripheral blood cells is mobilized; The mobilization of peripheral blood progenitor cell for collecting in the patient accepting marrow ablation (myeloablative) or bone marrow depression chemotherapy; And the Neutrophilic granulocytopenia after the induction/after treatment of acute myelogenous leukemia, fever, antibiotic usage, the minimizing of the time length of hospital care.Other state of an illness or obstacle can be treated with modification G-CSF, comprise asthma and allergic rhinitis.
As an other example, can be used for treating the disease such as nanism relevant to growth according to the human growth hormone (hGH) of method modification of the present invention, children and adult's is of short and small stature, emaciation/muscle consumption, general amyotrophy, and sex chromosomal abnormality (such as Turner's synodrome).Can comprise with the Other diseases that modification hGH treats: short bowel syndrome, lipodystrophy, osteoporosis, uremia (uraemaia), burn, female infertility, osteoproliferation, general diabetes, type ii diabetes, osteoarthritis, chronic obstructive pulmonary disease (COPD), and insomnia (insomia).And modification hGH can also be used for promoting various process, such as general tissue regeneration, osteanagenesis, and wound healing, or as vaccine adjuvant.
Therefore, in yet another aspect, the invention provides pharmaceutical composition.This pharmaceutical composition comprises the acceptable thinner of medicine and non-natural exists, water-soluble polymers, the covalent conjugates for the treatment of structure division or biomolecules and glycosylation or non-glycosylated peptide.This polymkeric substance, treatment connect base by structure division or biomolecules via intact glycosyl and put together in peptide, this intact glycosyl connect base between and be connected to peptide and polymkeric substance, treatment structure division or biomolecules with covalent linkage.
Pharmaceutical composition of the present invention is applicable to various drug delivery system.Be applicable to preparation of the present invention to find in Remington ' s Pharmaceutical Sciences, Mace PublishingCompany, Philadelphia, PA, 17th ed. (1985).For the summary of the method for drug conveying, see Langer, Science 249:1527-1533 (1990).
This pharmaceutical composition can be prepared for any suitable administering mode, such as, comprise local, mouth, nose, intravenously, encephalic, intraperitoneal, subcutaneous or intramuscular administration.For administered parenterally, such as subcutaneous injection, carrier preferably includes water, salt solution, alcohol, fat, wax or buffer reagent.For oral administration, any one or the solid carrier such as mannitol of above carrier can be used, lactose, starch, Magnesium Stearate, sodium sugar, talcum, Mierocrystalline cellulose, glucose, sucrose, and magnesiumcarbonate.Can also use biodegradable matrix, such as microballoon (such as polylactic-co-glycolic acid) is as the carrier of pharmaceutical composition of the present invention.The biodegradable microballoon be applicable to such as is disclosed in US patent Nos.4,897,268 and 5,075, in 109.
Usually, pharmaceutical composition subcutaneous or parenteral, such as intravenous administration.Therefore, the invention provides to comprise and be dissolved in or be suspended in acceptable carrier, preferred aqueous carrier, such as water, buffered water, salt solution, the composition for administered parenterally of the compound of PBS etc.Composition can also contain washing composition such as Tween 20 and Tween 80; Stablizer is N.F,USP MANNITOL such as, sorbyl alcohol, sucrose and trehalose; With sanitas such as EDTA and meta-cresol.Composition can containing the medicine acceptable auxiliary agent of simulation needed for physiological condition, such as pH regulator and buffer reagent, tensile conditioning agent, wetting agent, washing composition etc.
These compositions can carry out sterilizing by common sterilising technology, or can sterile filtration.Obtained aqueous solution can be packed, for direct use, or freeze-drying.Freeze-dried preparation merges with sterile aqueous carrier before administration.The pH of said preparation is generally 3-11, more preferably 5-9, most preferably 7-8.
In some embodiments, glycopeptide of the present invention can be incorporated into and be formed in the liposome that lipid formed by standard vesica.Multiple method is available for preparing liposome, such as, be described in the people such as Szoka, Ann.Rev.Biophys.Bioeng.9:467 (1980), U.S.Pat.Nos.4, and 235,871,4,501,728 and 4,837, in 028.Use being directed at (see such as US patent Nos.4,957,773 and 4,603,044) known in the art of the liposome of various directed agents (such as saliva acidic group galactoside of the present invention).
The standard method for directed agents being coupled to liposome can be adopted.These methods generally comprise the lipidic component that can activate and be used for connecting directed agents, such as phosphatidylethanolamine, or derivatize lipophilic compound, and such as lipid derivatize glycopeptide of the present invention is incorporated into liposome.
Directing Mechanism of V-notchf Withjn general requirement directed agents is positioned on the surface of liposome, make object construction part can with target compound, such as cell surface receptor interact.Carbohydrate of the present invention can be connected to lipid molecule (such as, being present in the alkylation or the acidylate that have long chain halides respectively or have the hydroxyl on the carbohydrate of lipid acid) before use method known to those skilled in the art forms liposome.Or, liposome can by formation film in by connection portion the mode be first incorporated in film make.This connection portion must have lipophilic portion, and it embeds and anchoring securely in film.It also must have reactive part, and it can chemical utilization on the aqueous surface of liposome.Selective reaction part, makes it chemically be suitable for forming stable chemical bond with the directed agents of adding afterwards or carbohydrate.In some cases, directly directed agents can be connected in connection molecule, but in most of the cases, be more suitable for use the 3rd molecule to be used as chemical bridge, therefore the connection molecule in film is connected with the directed agents of stretching out from vesicle surface solid or carbohydrate.
The compound prepared by method of the present invention can also be used as diagnostic reagent.Such as, the compound of mark can be used for locating the region being suspected to have inflammation in the patient of inflammation or metastases.For this reason, compound can be used 125i, 14c or tritiated.
Following examples provide for compound of the present invention and method are described, but do not limit required invention.
Embodiment
Following symbol is used in embodiment 1-3.Main PEG subunit is made up of four ethylene glycol unit.This molecule has the molecular weight of 194, is rounded to 200 in the drawings.Following symbol is used for representing main PEG subunit:
The binding substances of main PEG subunit is alternatively used in the molecular weight that rounds up shown between functionalized end as shown below and represents.
Following symbol is used for representing monofunctional methoxyl group PEG subunit:
Embodiment 1
The preparation of single dispersing PEG and their activated form
Single dispersing or unimodal molecular weight PEG is as follows prepares.By regulating the size of the fragment produced, prepare the PEG of any yardstick.Then by one end end-blocking of alkylation by glycol, and activate, for puting together in biological structure part such as albumen, sugar, lipid or Nucleotide.
Leavings group can be connected to main PEG subunit, to produce the main PEG of activation shown below.In the reaction, Q can be any leavings group compatible with chemical process of the present invention.Illustrative leaving groups comprises halogen, tresylate, tosylate and methanesulfonate.
After the main PEG of generation activation, as follows, this compound and main PEG subunit are reacted.This product is first-generation PEG continuation.
S-generation PEG continuation produces according to the mode same with the first-generation.
The generation as follows of third generation PEG continuation.
The generation as follows of forth generation PEG continuation.
PEG extends process and reacts to stop by allowing one of monofunctional structure division and difunctional compound.In the reaction, monofunctional structure division is any group compatible with chemical process of the present invention.Exemplary capping group comprises alkoxyl group-PEG and alkyl.
In following exemplary, leavings group is added in methoxyl group-PEG subunit.Then this molecule reacts with forth generation PEG continuation.
In another exemplary embodiment, methyl subunit is added on forth generation PEG continuation.
After end-blocking end, as follows carrying out activates for Bioconluaate the other end of PEG continuation.In the reaction, X is any leavings group that can form ester.Symbol X independently selected from imidazolyl, HOBt, HOAt, NHS, and p-nitrophenyl ester.
Finally, PEG extends that molecule is as follows puts together in biological structure part.
Embodiment 2
Do not require that activation single dispersing PEG must have the PEG subunit with the prolongation molecule similar number reacted with it.In an exemplary embodiment, the single dispersing PEG of activation has the PEG subunit than the prolongation molecule reacted with it more high number.In another exemplary shown below, activation single dispersing PEG has the PEG subunit than the prolongation molecule lesser number of reacting with it.
The end blocking method of these molecules is similar with method described in embodiment 1.
Embodiment 3
Excessive activated PEG subunit can be added, to produce single dispersing PEG shown below:
N is 1-100 or 1-20,000, depend on source
By changing the ratio of reagent, the alkali used, temperature, solvent and concentration, can regulate reaction, main molecules amount (n) needed for acquisition.
This method provide simple, quick, the effective means of the single dispersing PEG preparing any size.Purify and be simplified by the method, there is difference in (with therefore physics-chem characteristic) because the molecular weight of single dispersing PEG.This allows to use simple, standard purification techniques such as silica gel, anti-phase Mierocrystalline cellulose, membrane filtration (nanofiltration and ultrafiltration).The PEG glycol of purifying is then derivative turns to required any functionalized form.
Embodiment 4
The production of alkoxyl group PEG
General method shown below is used for preparing alkoxyl group PEG or other monofunctional PEG.
In the first embodiment, the generation as follows of Bifunctionalized PEG molecule is activated:
Wherein symbol n represents 1-100, the numerical value of 000.Symbol Q represents any leavings group compatible with chemical process of the present invention.Illustrative leaving groups comprises halogen, tresylate, tosylate and methanesulfonate.Any counter ion that sign X is compatible with leavings group.
This activation Bifunctionalized PEG molecule length for extending PEG molecule as follows:
Wherein symbol m represents 1-100, the numerical value of 000.
In second embodiment, as follows, extend monofunctional PEG, then activate, for biological structure moiety conjugation.
In a first step, monofunctional PEG carries out tosylation.
Wherein symbol n represents 1-100, the numerical value of 000.
In second step, extend monofunctional PEG:
Wherein symbol m represents 1-100, the numerical value of 000.
In last step, as follows, the monofunctional PEG compound activating of prolongation is used for and biological structure moiety conjugation.
Embodiment 5
For the preparation of other composition and the method for two feeler polymkeric substance
Other pair of feeler structure of the present invention has following general formula:
Wherein sign X OH, H, Q (activating group), and biological structure part, such as albumen, sugar, lipid, or Nucleotide.Symbol n represents the numerical value of 1-10.Term " polymer " " can be PEG, mPEG (methoxy poly (ethylene glycol)), PPG (polypropylene glycol), mPPG, polyglutamic acid, poly aspartic acid, poly(lactic acid), and Polysialic acid.
In an exemplary embodiment, two feeler structure has following general formula:
Wherein symbol m and n represents 1-10 independently, the numerical value of 000.Sign X OH, H, Q (activating group), and biological structure part, such as albumen, sugar, lipid or Nucleotide.
In another exemplary embodiment, two feeler structure has following formula:
Wherein symbol a and b represents the numerical value of 1-24 independently.Symbol m and o represents 1-10 independently, the numerical value of 000.Sign X OH, H, Q (activating group), and biological structure part, such as albumen, sugar, lipid or Nucleotide.
Although disclose the present invention with reference to specific embodiments, obviously, when not departing from true spirit of the present invention and scope, others skilled in the art can design other embodiment of the present invention and modification.
The all patents enumerated in this application, patent application and other publication are introduced for reference comprehensively.

Claims (9)

1. peptide, it has following general formula:
Wherein, WSP is water-soluble polymers, and
R 14be selected from OH, reactive functional groups, the group of sugary structure division or the one be connected in the group of carrier molecule.
2. peptide according to claim 1, wherein said water-soluble polymers comprises PEG.
3., according to the peptide of claim 1 or 2, it has following general formula:
Wherein:
M, n and t independently selected from integer 1-20,000.
4. according to the peptide of claim 1 or 2, wherein R 14comprise sugared structure division.
5. peptide according to claim 4, wherein said sugared structure division is nucleotide sugar.
6. peptide according to claim 4, wherein said sugared structure division is puted together in being selected from the one in the second peptide and lipid.
7. peptide according to claim 6, wherein said sugared structure division puts together the one in the amino acid being selected from described the second peptide and glycosyl residue.
8. peptide according to claim 7, wherein said sugared structure division is that the glycosyl between described peptide and described the second peptide connects base.
9. peptide according to claim 8, wherein said sugared structure division is that the intact glycosyl between described peptide and described the second peptide connects base.
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